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Developing a Method to Protect the Integrity
of Racing Using Targeted SRM: Detection and
Quantitation of rhEPO/DPO in Horse Plasma
Scott M. Peterman
1
, Cornelius Uboh
2
, Fuyu Guan
2,3
, Lawrence Soma
3
, Eric Birks
3
, and Jinwen Chen
3
1
Thermo Fisher Scientific, Somerset, NJ, USA;
2
Pennsylvania Equine Toxicology and Research Laboratory,West Chester, PA, USA;
3
University of Pennsylvania, Kennett Square, PA, USA
Key Words
• TSQ Quantum
Access
™
•
Heavy
Peptide
Labeled
Standards
• Proteotypic
Peptides
• SRM Method
• Targeted Protein
Analysis
Application
Note: 408
Overview
Purpose:
To develop a method for the detection and
confirmation of rhEPO/DPO in horse plasma using a
targeted protein assay and labeled internal standards.
Methods:
Combined immunoaffinity separation, enzymatic
digestion, and mass spectrometry has been used to confirm
the presence of rhEPO in horse plasma.
1
The use of an
SRM method for targeted protein detection enabled
measurements of retention times, ion ratios, and labeled
internal standards to confirm and quantify the presence
of rhEPO in horse plasma.
Results:
Using labeled internal standards, rhEPO was
detected, quantified and confirmed in administered horse
plasma 72 hours following administration, simulating
real world situations.
Introduction
Recombinant human erythropoietin (rhEPO)
2
and
Darbepoetin-alpha (DPO)
3
are genetically engineered
protein-based drugs used for the treatment of anemia by
stimulating red blood cell production. The ability of these
agents to stimulate red blood cell production has led to
use and abuse by human and equine athletes and, thus,
violates the rule of fair competition resulting in their
classification as banned substances by the horse racing
industry. In addition, continued administration to horses
can result in anemia.
3
Despite the negative aspects of rhEPO
for horses, a reliable, verifiable, and legally defensible
method for identification and confirmation of rhEPO/DPO
has been elusive due to the very low concentrations
administered. Sample collection is typically acquired only
after competition, which could be in excess of 72 hours
following administration. Testing of rhEPO/DPO is further
confounded by the complexity of the matrices in which the
drug is typically found–plasma and urine.
Methods
All experiments were performed using a Thermo Scientific
TSQ Quantum Access triple quadrupole mass spectrometer
equipped with a Thermo Scientific Surveyor
™
MS Pump
and MicroAS Autosampler (Thermo Fisher Scientific,
San Jose, CA) operated in mSRM mode monitoring six
diagnostic peptides that differentiate rhEPO and DPO
from equine EPO. (Scheme 1). In addition to the six
diagnostic peptides, four stable isotope labeled internal
standards for the T
4
, T
6
, T
11
, and T
17
rhEPO proteotypic
peptides were used for absolute quantification and addi-
tional confirmation of the presence of rhEPO/DPO (Thermo
Biopolymers, Thermo Fisher Scientific, Ulm, Germany).
Method development was performed using neat rhEPO/
DPO protein digests. (Amgen, Inc., Thousand Oaks, CA).
HPLC separations were achieved using a Hypersil
Biobasic
™
C18 100
×
0.5 mm column and a binary solvent
system consisting of A) 0.1% formic acid and B) MeCN
(0.1% formic acid). A gradient profile of 2-40% B in
12 minutes was used at 60 µL/min.
Sample preparation included immunoaffinity
separation using rabbit and mouse IgG antibodies linked
to magnetic beads. Following separation, the resulting
protein was filtered and enzymatically digested with an
enzymatic or proteolytic cleavage from which a set of
diagnostic peptides representing rhEPO/DPO was chosen
as candidate biomarkers for confirmation of the presence
of rhEPO/DPO in horse plasma.
1
Two different sets of samples were prepared and
analyzed. The first set was a controlled spiking experiment
in which a known quantity of rhEPO was spiked into 1 mL
of digested horse plasma to determine detection efficiency.
The second sample set was plasma extracted as a function
of time following rhEPO administration (iv) of 8000 IU.
The time points for extraction ranged from 0 hr to 72
hours. Each of the time point samples was spiked with
10 fmol/µL of the labeled peptide standards.
Results and Discussion
Scheme 1 shows the basis of identification for rhEPO/DPO
in equine plasma. The results of enzymatic digestion
produced multiple diagnostic markers that can be used
to increase the confidence of the presence of the foreign
substance in the equine athlete. In addition, the method
described enables detection of rhEPO or DPO due to the
conserved sequence for each protein over the targeted
peptides. Figure 1 shows summed SRM chromatograms
for (1A) DPO and (1B) rhEPO using the same SRM
transitions. Clearly, the retention times are closely
identical for both samples indicating the experimental
method is robust for either drug.