Analytical Chemistry Letters

ISSN: 2229-7928 (Print) 2230-7532 (Online) Journal homepage: http://www.tandfonline.com/loi/tacl20

Solid-Phase Extraction and HPLC-DAD for

Determination of Salbutamol in Urine Samples

Maha M. Abdelrahman

To cite this article: Maha M. Abdelrahman (2018) Solid-Phase Extraction and HPLC-DAD for
Determination of Salbutamol in Urine Samples, Analytical Chemistry Letters, 8:1, 35-45, DOI:
10.1080/22297928.2017.1396918

To link to this article: https://doi.org/10.1080/22297928.2017.1396918

Published online: 15 Mar 2018.

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 TACL 8 (1) 2018 pp 35 - 45 35
ISSN Print: 2229-7928
ISSN Online: 2230-7532

Solid-Phase Extraction and HPLC-DAD for

Determination of Salbutamol in Urine Samples

Maha M. Abdelrahman
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef
University, Alshaheed Shehata Ahmad Hegazy St., 62514, Beni-Suef, Egypt
Received 24 January 2017; accepted in revised form 14 October 2017

Abstract: Validation of a solid-phase extraction and a high-performance liquid chromatographic assay

for determination of Salbutamol in urine samples is described. HPLC-DAD method was established for
determination of Salbutamol in urine samples following inhaled administration by 0.5 and 24 h after cleanup
procedure. Agilent ODS 5 μm, 4.6 x 250 mm, C-18 stationary phase with a mobile phase consisting of a mixture
of acetonitrile and water (adjusted to pH = 3 using orthophosphoric acid) (90: 10, v/v) at a flow rate 1 mLD min
with detection at 220 nm and Bambuterol HCl as an internal standard were used for development of the method.
Solid phase extraction was performed using Oasis MCX cartridge for both un-metabolized Salbutamol (excreted
after 0.5 h) and metabolized Salbutamol (excreted after 24 h) with mean percentage recoveries of 92.24 % and
91.52 %, respectively. The method described permits the detection of Salbutamol in human urine at concentration
as low as 0.15 μg/mL. The proposed method was successfully applied for determination of Salbutamol in urine
samples after inhalation of a metered dose inhaler (MDI) of Ventolin® to healthy volunteer after 0.5 and 24 h. The
proposed method can be used for pharmacokinetic study of Salbutamol and determination of its relative
deposition in the lung.

Key words: Salbutamol; HPLC; solid phase extraction; urine; method validation.

Introduction from the gastrointestinal tract. It is subject to first-

Salbutamol (or albuterol) chemically is (1RS)-
2-[(1,1-Dimethylethyl)amino]-1-[4-hydroxy-3-
(hydroxymethyl)phenyl]ethanol 1, 2. It is a short
acting β2-adrenergic receptor agonist that is used
as a bronchodilator in the treatment of bronchial
asthma. It was first introduced in 1970 3, 4. SAL is
one of the most important medications needed in
a basic health system listed by the World Health
Organization’s List of Essential Medicines 5.
Salbutamol (SAL) is usually taken by the in-
haled route for direct effect on bronchial smooth
muscle. This is mostly accomplished through a
metered-dose inhaler (MDI), nebulizer, or other
proper devices, SAL is usually delivered as
Salbutamol sulfate. Salbutamol is readily absorbed
pass metabolism in the liver and possibly in the
gut wall; the main metabolite is an inactive sul-
fate conjugate. Salbutamol is rapidly excreted in
the urine as metabolites and unchanged drug 6.
Like all drugs delivered by metered dose inhal-
ers (MDIs) only a small proportion of the actu-
ated dose reaches the lungs following inhalation.
The majority of the dose is deposited in the
oropharyngeal region and is subsequently swal-
lowed. Therefore, pharmacokinetic methods to
assess the pulmonary deposition of Salbutamol are
difficult due to the problems associated with the
swallowed part 7.
After oral administration of SAL, negligible
amount of salbutamol is excreted during 0.5 h post

*Corresponding author (Maha M. Abdelrahman)

E-mail: < maha_m_abdelrahman@yahoo.com > © 2018, Har Krishan Bhalla & Sons
 Maha M. Abdelrahman / TACL 8 (1) 2018 35 - 45
dose while considerably greater amount is excreted
0.5 h post inhalation, although similar amounts
were excreted over the 24 h period post inhaled
dosing. Therefore the amount of the drug in the
urine sample taken 0.5 h post inhalation can be
used as a representative of the amount of drug
delivered to the lungs (relative lung bioavailability
of SAL following inhalation) while the 24 h re-
covery can reflect the total SAL delivered to the
body following inhalation (relative systemic
bioavail-ability of SAL following inhalation) 7.
Different HPLC methods were described in lit-
erature for determination of SAL alone or in com-
bination with other components 8-15. Several meth-
ods were developed for SAL detection in biologi-
cal fluids 16-28. Methods reported for detection of
SAL in biological fluids used either fluorescence
16-20
or MS/MS 21-24 or electrochemical 25-28 de-
tections. Despite of the good specificity and sen-
sitivity of most of these methods, they are costly,
complicated for routine clinical studies and requires
sophisticated instruments which are not available
in most laboratories.
For determination of pharmaceuticals in biologi-
cal samples, analyst encounters different chal-
lenges, including biological matrices, low analyte
concentration and other possible interfering com-
ponents. In this work a HPLC-DAD method was
developed for determination of Salbutamol in urine
sample, although HPLC-DAD has the advantage
of being available over other chromatographic in-
struments (LC/MS LC/fluorescence and CE) but
it suffers low sensitivity. To conquer these cir-
cumstances; extracted urine samples were in-
creased by 10 folds (10 mL urine samples were
used), isolated samples after SPE were concen-
trated by 10 folds and injection volume was in-
creased to 50 μg/mL without affecting the re-
quired standards of method validation parameters.
For the first time a HPLC-DAD method is de-
scribed for selective determination of un-metabo-
lized and metabolized SAL in urine samples with
simple pretreatment SPE procedure within short
analysis time. The aim of the work is to develop a
sensitive, robust and reliable HPLC-DAD assay
for the determination of Salbutamol concentra-
tion in urine samples following inhaled adminis-
tration. Hence, this determination could be used
36
to study the relative deposition of Salbutamol in
the lung.
Experimental
Apparatus
HPLC (Agilent 1260 Infinity, Germany) instru-
ment was equipped with Agilent 1260 Infinity pre-
parative pump (G1361A), Agilent 1260 Infinity Di-
ode array detector VL (G131SD), Agilent 1260
Infinity Thermostated column compartment
(G1316A) and Agilent 1260 Infinity preparative
Autosampler (G2260A). Separation and
quantitation was performed on ZORBAX Eclipse
Plus C18 column (250 × 4.6 mm i.d, 5 μm particle
size (USA).
Reagents and materials
Salbutamol sulfate and Bambuterol HCl were
given as a gift from Alexandria Co. for Pharma-
ceuticals and Chemical Industries, Alexandria,
Egypt.
Acetonitrile and methanol (HPLC grade) were
supplied by (Chromasolv®, Sigma-Aldrich Chemie
GmbH, Germany)
Deionized water (SEDICO pharmaceuticals
Co., Cairo, Egypt).
Triethylamine (TEA) was obtained from Riedel-
deHäen (Sleeze, Germany), orthophosphoric acid
(85 %), ammonia solution (Specific gravity 0.91,
33 %) and hydrochloric acid (El-Nasr Pharma-
ceutical Chemicals Co., Abu-Zabaal, Cairo,
Egypt).
Oasis MCX 1 cc Vac cartridge, 30 mg sorbent,
30 μm particle size (International Sorbent tech-
nology, UK).
Standard solutions
Aqueous stock solution of Salbutamol 500 μg/
mL (w/v) equivalent to 1205.5 μg/mL Salbutamol
sulphate was prepared in acetonitrile and stored
at -20oC. Two series of working standards (for
both un-metabolized and metabolized SAL) were
prepared by diluting stock solution of SAL using
acetonitrile: H2O (90:10, v/v) to obtain 50 μg/mL
solutions then stored at 5oC.
Aqueous stock solution of Bambuterol HCl 500
μg/mL (w/v) was prepared in acetonitrile and
stored at -20oC. Working standard solution was
 Maha M. Abdelrahman / TACL 8 (1) 2018 35 - 45
prepared by diluting its stock solution using ac-
etonitrile: H2O (90:10, v/v) to obtain 50 μg/mL so-
lution then stored at 50C.
Chromatographic conditions
The HPLC stationary phase was a Zorbax ODS
5 μm, 4.6 x 250 mm, C-18 (Agilent technology,
Agilent, UK). The mobile phase was a mixture of
acetonitrile and water (adjusted to pH = 3 using
orthophosphoric acid) (90: 10, v/v). The mobile
phase was filtered through a 45 mm membrane
filter (Millipore). A constant flow rate of 1 mL/
min was used and photodiode array detection was
set at 220 nm. An operating temperature of 25oC
was used throughout the analysis and the injec-
tion volume was 50 μg/mL.
Urine samples preparation
Two series of urine samples were prepared with
which pooled free urine (obtained from 5 healthy
volunteers) were spiked with differernt concen-
tration of SAL for both un-metabolized and me-
tabolized determination’s procedures. The ob-
tained concentrations of the respective solutions
were in the range of 0.5-50 μg/mL. Each con-
centration was prepared in triplicates. Non-spiked
samples were also prepared in order to correctly
assess recoveries.
SPE extraction procedures
SPE of un-metabolized SAL (pre-hydrolysis)
Solid phase extraction of un-metabolized SAL
was carried out using mixed mode cationic-ex-
change Oasis MCX cartridge connected to a res-
ervoir. The prepared spiked urine samples (10 mL)
were acidified with 5 mL 0.1 N HCl solution and
0.1 mL of Bambuterol working solution (as inter-
nal standard) was added then mixed well. Each
Oasis MCX cartridge was conditioned with 5 mL
methanol then equilibrated with 5 mL of deion-
ized water followed by loading of the pre-treated
urine samples. Then 5 mL of 0.5 % methanol in
0.1 N HCl was added to the cartridge. The car-
tridges were then dried for 2 min using a low
vacuum. After passing 5 mL methanol through
the cartridges they were dried for 2 min using a
full vacuum. The cartridges were washed with 5
mL of 2.5 % TEA in methanol and then dried again
for ~5 min under full vacuum. The analytes (SAL
37
and internal standard) were eluted with 5 mL of 5
% NH4OH in methanol into glass test tubes. Af-
ter evaporation to dryness using a nitrogen stream
at 650C for 15 min, the residue was reconstituted
in 1 mL of the mobile phase and then injected into
the HPLC system. Blank urine (free from SAL
and Bambuterol) was treated similarly.
SPE of metabolized SAL (post-hydrolysis)
The prepared spiked urine samples (10 mL)
were first hydrolyzed to convert all metabolized
SAL to the free SAL. 10 mL urine samples were
mixed with 2 mL 0.5 N HCl in glass test tubes
and covered with aluminum foil then inserted into
a boiling water bath for 5 min, then cooled. 0.1
mL of Bambuterol working solution (as internal
standard) was added, then mixed well. The pre-
treated urine samples after acid hydrolysis were
then loaded on Oasis MCX cartridges and the pro-
cedure of un-metabolized SAL was then followed.
Blank urine (free from SAL and Bambuterol) was
treated similarly.
Validation of the method
The optimized method was validated by ana-
lyzing different concentrations of SAL in urine ma-
trix for pre- and post-hydrolysis procedures in the
range 0.5-50 μg/mL. Each concentration was ana-
lyzed three times. Validation parameters such as
linearity, intra-day and inter-day precision (%
RSD), recovery (R), accuracy, limit of detection
and limit of quantification were established. Re-
covery of SAL was determined by repeated SPE
procedures (n=3) of three urinary SAL concen-
trations (0.5, 10 and 20 μg/mL) for both pre- and
post-hydrolysis urine samples. The relative peak
areas of urine extracts of SAL were compared to
the peak areas obtained with the direct injection
of SAL aqueous standards in order to provide an
estimate of the extraction recovery.
Validity of the developed SPE procedure was
further examined by applying to a volunteer’s 0.5-
24 h urine sample after inhalation of twelve puffs
of SAL from a MDI (Ventolin® GlaxoSmithKline,
UK).
Real sample application (volunteer study)
Urine samples were collected from a single vol-
unteer who inhaled twelve puffs from a Ventolin®
 Maha M. Abdelrahman / TACL 8 (1) 2018 35 - 45
metered dose inhaler (100 μg Salbutamol sulfate
per dose), each inhaled dose was separated by
30 seconds. Immediately prior to each study dose,
subjects emptied their bladder. Urine was then
collected at 0.5 and 24 h post-dose. Samples were
frozen at -200C prior to extraction and HPLC
analysis.
Results
Optimization of the chromatographic method
Different mobile phases were tried to obtain
good resolution with reasonable retention time.
During development of the chromatographic pro-
cedure blank urine samples were injected to
achieve the required separation between urine
peaks and SAL peak for both pre- and post-hy-
drolysis conditions. Where optimum separation of
SAL peak with a good retention time was attained
using a mixture of acetonitrile and water (adjusted
to pH=3 using orthophosphoric acid) (90: 10, v/
v). Acidic pH was essential to provide good sepa-
ration of SAL and urine peaks and improved peaks
shape.
It was difficult to find a compound to be used
as an internal standard. Different compounds
were investigated such as, atenolol, hydrochlo-
rothiazide, metronidazole, clopidogrel, carvedilol,
methocarbamol, miconazole, xipamide, orphen-
adrine and chlorzoxazone. But these drugs either
have long retention time or eluted early and inter-
fered with SAL or urine peaks. Finally Bambuterol
HCl was found to be the most appropriate inter-
nal standard under the separation condition.
The diode array detector (DAD) was adjusted
at different detection wavelengths (215, 220, 230
and 276 nm) in order to achieve optimum sensi-
tivity and selectivity. It was found that SAL and
Bambuterol attained higher sensitivity at 220 nm.
Evaluation of the extraction procedures
For the biological samples clean-up, some of
the HPLC methods used methanol or acetonitrile
to precipitate the protein. The advantages of this
procedure are speed and ease, but the disadvan-
tage is incomplete precipitation of the plasma pro-
tein, also the final organic phase contains not only
endogenous substances that are soluble in the sol-
vent but also some water-soluble compounds
which badly affect separation efficiency and may
38
damage HPLC column 29. SPE technique for iso-
lation of biological samples has been widely used
for extraction of different pharmaceuticals either
from plasma or urine samples 30-36.
Different cleanup procedures were adopted to
attain better recovery of SAL. Liquid-liquid ex-
traction (LLE) technique were tested using hex-
ane, CH2Cl2 and ethyl acetate and the results were
compared with that obtained from solid phase ex-
traction (SPE) using Oasis WAX, Oasis MCX,
Isolute HCX, Dionex SolEx C18) as sorbents.
Effective isolation and higher % recovery of SAL
was obtained upon using Oasis MCX cartridge,
as given in Table 1.
The isolation protocol adopted on Oasis MCX
cartridge was modified and optimized to attain
highest purification and recovery of SAL with
minimum steps. Also, the purification steps were
designed to extract un-metabolized SAL and me-
tabolized SAL using the same type of cartridge
for simplicity. The clean-up stage using Oasis HCX
cartridges isolated SAL and internal standard from
urinary endogenous substances that might inter-
fere with the assay gave a highly, reproducible
absolute recovery (> 90 %), as given in Table 2.
Mixed mode cation-exchange cartridge (Oasis
MCX) was found to be much more efficient (re-
covery = 92.24 and 91.52 %). This could be at-
tributed to that SAL is a basic compound (pKa=
9.4) and a strong cation exchange sorbent allows
for complete retention of basic drugs. The purifi-
cation protocol adopted for the selected sorbent
is based on that, basic drugs to be retained on
cartridge, pH is first adjusted to be acidic (using
HCl solution) for washing steps of interfering en-
dogenous compounds present in urine matrix. At
this acidic pH SAL will be in cationic form so
strongly attached to sorbent. After washing steps,
pH is adjusted to be basic (using NH4OH solu-
tion) at which SAL is uncharged so eluted from
the cartridge.
Finally, the chromatographic separation of SAL
and internal standard was carried out using a mix-
ture of acetonitrile and water (adjusted to pH=3
using orthophosphoric acid) (90: 10, v/v) delivered
at 1 mL/min at 250C and detection at 220 nm.
Fig. 1 shows the separated chromatograms of
SAL and internal standard for pre- and post-hy-
drolysis conditions and volunteer application.
 Table 1. Extraction procedures of Salbutamol from urine samples

Solid Phase Extraction (SPE)

Cartridge Conditioning Equilibrium Loading Washing 1 Washing 2 Elution % Recovery

Oasis WAX 5 mL methanol 5 mL H2O Urine acidified 5 mL 2 % 5 mL methanol 5 mL 5 % 54.84

with 5 mL 0.1 HCOOH NH4OH in
N HCl methanol
Oasis MCX 5 mL methanol 5 mL H2O Urine acidified 5 mL 5 % 5 mL methanol 5 mL 5 % 92.24
with 5 mL 0.1 methanol in then 5 mL 2.5 % NH4OH in
N HCl 0.1 N HCl TEA in methanol methanol
Isolute HCX 5 mL methanol 5 mL 0.05 M Urine + 5 mL 5 mL 0.05 M 5 mL 1 M 5 % NH3 78.72
ammonium 0.05 M ammonium ammonium acetic acid in methanol
acetate acetate buffer acetate then 5 mL
buffer (pH 6) (pH 6) buffer (pH 6) methanol
Dionex SolEx 5 mL methanol 5 mL H2O Urine acidified l 5 mL KH2PO4 6 mL ACN: 5 mL ACN 68.5
C18 with 5 mL buffer (pH=5) KH 2PO4 buffer
0.1 N HC (pH=5) (10:90, v/v)

Liquid-Liquid extraction (LLE)

Maha M. Abdelrahman / TACL 8 (1) 2018 35 - 45

Solvent Hexane CH 2Cl 2 Ethyl acetate

% Recovery 54.8 37.2 53.8

39
 Maha M. Abdelrahman / TACL 8 (1) 2018 35 - 45

Fig. 1. Chromatograms obtained from the analysis of (a) an extracted blank urine sample (pre-hydrolysis) (b) an extracted urine sample
containing 2 μg/mL Salbutamol and 5 μg/mL Bambuterol (pre-hydrolysis) (c) a volunteer urine sample 0-0.5 h post inhalation (pre-
hydrolysis) (d) an extracted blank urine sample (post-hydrolysis) (e) an extracted urine sample containing 2 μg/mL Salbutamol
and 5 μg/mL Bambuterol (post-hydrolysis) (f) the same volunteer urine sample 0.5-24 h post inhalation (post-hydrolysis)
40
 Maha M. Abdelrahman / TACL 8 (1) 2018 35 - 45
Method validation
The HPLC-DAD method for determining un-
metabolized and metabolized SAL in urine samples
was validated. The validation parameters obtained
are listed in Table 2. Method linearity was estab-
lished on the basis of six different concentrations
of SAL in urine matrix analyzed with three repli-
cates. The method was linear over a wide range
of concentrations; 0.5-50 μg/mL with correlation
coefficients 0.9981 and 0.9973 for both pre- and
post-hydrolysis samples, respectively. Accuracy
of the method was determined by assessing the
agreement between the measured and known con-
centrations of the samples. This value ranged from
96.47-104.07 % for pre-hydrolysis samples and
from 94.78-103.56 % for post-hydrolysis samples.
The intra-day precision of the method was deter-
mined by calculating the relative standard devia-
tion (% RSD) for the repeated measurements of
SAL on three successive times in the same day
and inter-day precision was performed using the
same concentrations but on three successive days
for pre- and post-hydrolysis procedures, as pre-
sented in Table 3. The assay has acceptable lim-
its for both accuracy and precision and has been
successfully used to analyze samples from this
study, and other subsequent clinical studies. The
lower limit of detection (LOD) fully confirmed
the usefulness of this method for the analysis of
Table 2. Regression and validation parameters of the proposed
HPLC method for determination of Salbutamol
41
SAL in urine samples. The LOD and LOQ for
urine sample were 0.15 and 0.5 μg/mL for pre-
hydrolysis and 0.17 and 0.5 μg/mL for post-hy-
drolysis conditions, respectively, Table 2.
Discussion
Salbutamol is frequently used for treatment of
asthma. SAL may be restricted in certain sports,
as it is considered to be a member of a prohibited
group (β-2 agonists). Urinary SAL concentrations
are frequently measured in competitive sports pro-
grams, for which a level in excess of 1 μg/mL is
considered to represent abuse 37-39. From the ana-
lytical point of view, it is a matter to exploit a se-
lective, accurate and simple method to allow de-
tection of SAL in urine samples either after in-
haled or oral administration.
The urinary excretion of individuals is difficult
to control over the collection time. This is particu-
larly important when patients are unable to inhale
SAL dose properly and completely from a MDI
resulting low levels excreted in urine. In such situ-
ation, a large output of urinary volume may de-
mand the use of more than the usual 1 mL of
urine sample for the SPE to ensure consistent
chromatographic response. Large volume of urine
sample (10 mL) allow proper measurement of
SAL concentration to overcome low sensitivity
of DAD detector.

Parameters Pre-hydrolysis (0.5 h) Post-hydrolysis (24 h)

Linearity range (μg/mL) 0.5 - 50

Slope 0.2232 0.1870
Intercept - 0.0614 0.1370
Correlation coefficient (r) 0.9981 0.9973
Accuracy (mean ± SD) 99.66 ± 2.87 98.65 ± 3.74
% Recovery 92.24 91.52
LOD (μg/mL) 0.15 0.17
LOQ (μg/mL) 0.50 0.50
Robustness (% RSD)
Percentage of organic 2.05 3.26
solvent (acetonitrile, 90 % ± 2)
pH of the mobile phase (3 ± 0.02) 1.46 1.58
Mobile phase flow rate 1.31 2.34
(1 mL/min ± 0.1)
 Maha M. Abdelrahman / TACL 8 (1) 2018 35 - 45 42
Table 3. Precision of Salbutamol using the developed HPLC method in urine

SAL μg/mL) % recovery* Mean* (μ

Mean* (μ μg/mL) % recovery*
Concentration of measured of measured of measured of measured
μg/mL)
(μ Concentration Concentration Concentration Concentration
Intra-day precision a
Pre-hydrolysis of SAL Post-hydrolysis of SAL

0.50 0.475 95.00 ± 3.51 0.526 105.20 ± 2.75

5.00 5.213 104.26 ± 2.33 4.797 95.94 ± 3.84
10.00 9.766 97.66 ± 2.75 10.36 103.60 ± 4.10
Mean ± % RSD 98.97 ± 4.77 101.58 ± 4.95
Inter-day precision b
0.50 0.521 104.20 ± 4.23 0.476 95.20 ± 3.47
5.00 5.201 104.02 ± 5.52 4.806 96.12 ± 2.72
10.00 9.802 98.02 ± 4.82 10.39 103.90 ± 3.88
Mean ± % RSD 102.08 ± 3.52 98.41 ± 4.78

a
The intraday (n = 9), average of three different concentrations repeated three times within day
b
The interday (n = 9), average of three different concentrations repeated three times in three successive
days
Mazher and Chrystyn 17 developed a sensitive
HPLC method for determination of SAL with fluo-
rescence detection but with long analysis time (re-
tention of SAL, 24 min) and used complicated
cleanup SPE steps and mobile phase and two
types of cartridges for un-metabolized and me-
tabolized SAL. While Zhang et al. 22 established
LC-MS/MS method for SAL determination based
on LLE of urine samples and didn’t take in con-
sideration determination of metabolized SAL. In
this work, a HPLC-DAD method was introduced
for selective assay of SAL in urine samples ei-
ther un-metabolized (excreted after 0.5 h) or
metabolized (excreted during 24 h). The suggested
method has the advantages over other published
methods in that it requires simple pre-treatment
steps for urine samples and short run time of
analysis (9 min) with satisfactory sensitivity and
used the same type of cartridge for determination
of both un-metabolized and metabolized SAL.
In comparison to other methods described in
the literature for determination of SAL in urine
samples, the described method yielded a compa-
rable recovery. SPE procedure and HPLC mo-
bile phase is simpler than that developed by pre-
vious HPLC methods. It was found that using the
same cartridge type for both pre- and post-hy-
drolysis purification steps is effective and good
recoveries were obtained.
Conclusion
Few RP-HPLC methods have been reported
for the detection of Salbutamol in urine matrix.
Some of the published methods require sophisti-
cated techniques, not suitable for determination
of both un-metabolized and metabolized
Salbutamol and need complicated purification
steps. The aim of this study was therefore to over-
come these difficulties. Hence this is the first re-
port on an extraction method for the detection of
Salbutamol in urine samples by HPLC-DAD
method with a reasonably short run time and simple
SPE isolation procedure. The proposed method
has been developed, optimized and validated us-
ing Bambuterol HCl as the internal standard and
it has been used for other subsequent clinical stud-
ies and can be applied for determination of
Salbutamol after oral administration.
Acknowledgment
I would like to acknowledge Dr. Mohamed E.
Abdelrahim; head of clinical pharmacy depart-
ment, faculty of pharmacy, Beni-Suef University
for his support and advice to perform this work.
 Maha M. Abdelrahman / TACL 8 (1) 2018 35 - 45 43
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