Page 343 - CRFTApplication Compendium_090613

Improved Signal-to-Noise Ratio in the
Antidoping Analysis of Clenbuterol in Urine
Using LC–FAIMS–H-SRM
James Kapron1 and Rohan Thakur2
1Thermo Fisher Scientific, Ottawa, Canada; 2Thermo Fisher Scientific, San Jose, CA, USA
Application
Note: 394
Key Words
• TSQ Quantum
Ultra ™
• Surveyor™ HPLC
• Drug Screening
• FAIMS Technology
• Improved
Selectivity
Introduction
Clenbuterol is a beta-2 agonist drug with anabolic
properties that is commonly used as a bronchodilator in
veterinary medicine (Figure 1). Its use in humans has been
banned in many countries, including the United States,
because of serious cardiovascular and pulmonary side
effects. The short-term effects of clenbuterol are similar
to stimulant drugs like amphetamine or ephedrine and
include increased heart rate, temperature, and blood
pressure. Clenbuterol also increases lean muscle mass
while reducing fat deposition. Some athletes and body-
builders use the drug for these thermogenic and anti-
catabolic effects, and as a result clenbuterol has been
banned by the World Anti-Doping Agency (WADA).
Doping control laboratories must therefore routinely
monitor clenbuterol in biological samples. Although
the analysis of clenbuterol by LC-MS/MS is selective,
endogenous matrix interferences often produce a high
chemical background. Reducing the chemical background
leads to improved detection of clenbuterol for monitoring
purposes. The selectivity of the LC-MS/MS method is
increased with the addition of both the gas-phase selec-
tivity of FAIMS (high-Field Asymmetric waveform Ion
Mobility Spectrometry) and H-SRM (Highly-Selective
Reaction Monitoring).
FAIMS and H-SRM work together to increase assay
selectivity. In the interface between the ion source and the
mass spectrometer, FAIMS selects which ions are allowed
into the vacuum region. By applying alternating low and
high electric fields, interferences are filtered out. The result
is LC-MS/MS chromatograms with reduced chemical
background and endogenous interferences. H-SRM, in
turn, provides higher analyte selectivity through improved
mass resolution of the precursor ion with Q1 while
maintaining high transmission efficiency. The net result
is cleaner chromatograms and more reliable results.
Goal
To improve the selectivity of an LC-MS/MS method
for the analysis of clenbuterol in urine using FAIMS
in combination with H-SRM.
Experimental Conditions
Sample Preparation
Standard calibration samples of human urine fortified
with clenbuterol were prepared at the following nine
concentrations: 0.5, 1, 1.5, 2.5, 5, 10, 25, 50, and
100 ng/mL. Quality control samples of human urine
fortified with clenbuterol were prepared at the following
four concentrations: 0.5, 1.5, 50, and 100 ng/mL. Blank
samples of human urine without the reference standard
were also prepared.
To prepare each sample for analysis, 75 µL of sample
was added to 225 µL of water. After mixing, 10 µL was
injected. No internal standard was used.
HPLC
HPLC analysis was performed using the Surveyor HPLC
System (Thermo Scientific, San Jose, CA). The 10 µL
samples were injected directly onto a 4.6 ! 50 mm
polar RP column. The mobile phase was acidified
acetonitrile/water delivered at a flow rate of 400 µL/min.
The gradient is described in Table 1.
Time (min.) %B
0 10
2 67
2.2 67
2.5 10
3 10
Table 1: Gradient profile
Mass Spectrometry
MS analysis was carried out on a TSQ Quantum Ultra
triple quadrupole mass spectrometer with a heated
electrospray ionization (H-ESI) probe (Thermo Scientific,
San Jose, CA). The MS and FAIMS conditions were
as follows:
Mass Spectrometry Conditions
Ion source polarity: Positive ion mode heated ESI
Spray voltage: 3500 V
Vaporizer temperature: 400°C
Sheath gas pressure (N 2): 100 units
Auxiliary gas pressure (N 2): 60 units
Ion transfer tube temperature: 300°C
Scan Type: SRM and H-SRM
Figure 1: Structure of Clenbuterol
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