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Rapid Quantitative and Confirmational
Screening for Drugs in Race Horse Urine
by ESI-LC-MS/MS and MS
3
Patrick Russell,
1
Paul Steinberg,
2
Mary L. Blackburn,
2
Diane Cho,
2
Robert Heather
2
1
University of Florida, Racing Laboratory, Gainesville, Florida;
2
Thermo Fisher Scientific, San Jose, California
Application
Note: 350
Key Words
• LTQ
™
• Confirmation
• Drug Screening
• MS
3
• Quantitation
Introduction
Drugs of abuse in the horse racing industry encompass
a variety of chemical classes and are typically analyzed
from a complex urine matrix. These factors render
the rapid and effective diagnostic screening of these
drugs at low levels difficult. Traditionally, quantitation
has been performed by triple quadrupole mass
spectrometry using reaction monitoring (SRM) mode.
However, this method does not monitor structurally
diagnostic fragmentation. Thus, a second step involving
derivatization and GC/MS confirmation was required.
Goal
To develop a simple and fast, yet rugged LC/MS based
method capable of simultaneous qualitative and quantita-
tive analysis. We have evaluated the application of the
Thermo Scientific LTQ linear ion trap mass spectrometer
for providing low levels of detection, good reproducibility,
and wide linear dynamic range required for reliable quan-
titation, with simultaneous structural confirmation using
diagnostic full-scan MS/MS or MS
3
mass spectrometry.
Experimental Conditions
Sample Preparation
Standards and Unknowns: Standards of the compounds
listed in Table 1 were prepared neat and in urine.
Urine standards and unknowns were spiked, dried,
and reconstituted with 90% water and 10% acetonitrile
with 0.1% acetic acid. Typical Instrument Setup settings
are shown in Figure 1.
HPLC
HPLC System: Thermo Scientific Surveyor
™
LC system
Column: Thermo Scientific BETASIL
™
C18, 3 µm,
100
×
2.1 mm
Flow Rate: 350 µL/min
Injection Volume: 10 µL (full loop)
Mobile Phase: (A) Water with 0.1% acetic acid
(B) Acetonitrile with 0.1% acetic acid
Gradient: 92% A to 90% B.
MS
Mass Spectrometry: Thermo Scientific LTQ linear ion trap
mass spectrometer
API Source: Thermo Scientific Ion Max
™
source with
electrospray ionization (ESI) probe
Ion Transfer Capillary: 220°C; Sheath Gas: 30 units
Auxiliary Gas: 0 units; Sweep Gas: 20 units
Spray Voltage: 4.5 kV; Isolation Width: 3 amu
Normalized Collision Energy™: 28%
WideBand Activation
™
: Applied as needed (see Table 1)
Ion Polarity Mode: positive or negative (see Table 1)
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