Abstracts of the Scientific Posters, 2013 AACC Annual Meeting ...

Mass Spectrometry Applications
Tuesday, July 30, 9:30 am – 5:00 pm
Objective: The goal of this study was to verify that tryptic peptides could be used to
quantitate Infliximab and to differentiate the drug from other human immunoglobulins
in serum.
Methods: A list of tryptic peptides unique to the heavy and light chain variable
regions were predicted by in silico digestion of Infliximab variable region sequences
found in the IMGT database (http://www.imgt.org/3Dstructure-DB). Infliximab
(RemicadeTM, Janssen Biotech, Inc.) was reconstituted to 10 mg/mL in 50
mM ammonium bicarbonate, reduced, alkylated and digested with trypsin (1:20
enzyme:substrate) at 37oC for 4 hours. Digests were analyzed by IDA LC-ESI-Q-
TOFMS; the most abundant peptides matching the in silico list were chosen for
subsequent studies. Quantitation of Infliximab was accomplished using standard SRM
analysis on an ABSciex API 5000 using pooled human serum from healthy controls
or 50 mM ammonium bicarbonate, each spiked with Infliximab. A 9-point standard
curve was generated [blank, 0.25, 0.5, 1, 2, 5, 10, 20 and 50 ug/mL]. A known
concentration of purified horse IgG (200 ug/mL) with a unique non-human constant
region peptide was added to each sample as a pre-analytical digestion control along
with stable isotope-labeled peptide internal standards to monitor HPLC retention
times. Samples were processed to remove non-immunoglobulin proteins using the
Melon Gel purification kit (Pierce, Rockford, IL), followed by trypsin digestion.
Peptides were separated on reverse-phase C18 liquid chromatography (Atlantis T3
3x100 mm) and subjected to MS/MS.
Results: Tryptic peptides unique to Infliximab were identified for both the heavy
and light chains and blasted against a human database; no significant cross-matches
were identified. Heavy and light chain peptides were quantitated in buffer with a
coefficient of variation (CV) of 11% and 5%, respectively, measured by the analyte/
horse IgG peak-area ratio at 20 ug/mL Infliximab. Limit of detection was 0.25ug/
mL, with a linear dilution response between 100-0.25 ug/mL (R2>0.99) for both
heavy and light chain peptides. After spiking Infliximab into the serum matrix,
CVs of 20% were obtained for the heavy and light chain peptides, with the lowest
detectable concentration at 5.0ug/mL. The dilution response was linear from 50-2 ug/
mL (R2>0.99) for both peptides.
Conclusions: While sensitivity and precision merit further development and studies
with samples from patients taking Infliximab are warranted, we have demonstrated the
ability to quantitate Infliximab using variable region peptides by LC-MS/MS in the
presence of other human immunoglobulins. This analytical approach has the potential
to be quickly adaptable to other drugs in this class and to significantly improve patient
care.
A-164
A Liquid Chromatography Tandem Mass Spectrometry Method
for Measurement of Erythrocyte 6-Mercaptopurine Metabolites in
Patients on Thiopurine Therapy
I. Tsilioni, T. Law, J. Dunn, R. W. A. Peake, M. D. Kellogg. Boston
Children’s Hospital, Boston, MA
Background: Thiopurine drugs such as 6-mercaptopurine (6-MP) and azathioprine
are used as immunosuppressive agents for the treatment of inflammatory
bowel disease (IBD). Measurement of erythrocyte 6-thioguanine (6-TG) and
6-methylmercaptopurine (6-MMP) concentrations is advocated for the purpose
of obtaining baseline levels prior to commencement of therapy and for monitoring
levels in patients receiving treatment. We describe a liquid chromatography tandem
mass spectrometry (LC-MS/MS) method for measurement of 6-TG and 6-MMP in
erythrocytes.
Materials and Methods: 6-thioguanine nucleotides (6-TGNs) and
6-methylmercaptopurine nucleotides (6-MMPNs) were extracted from 100 uL
of erythrocytes with perchloric acid and hydrolyzed to form 6-TG and 6-MMP
respectively. Liquid chromatography was carried out using a Shimadzu SIL20AC
autosampler with 20AD pumps (Shimadzu Corporation, Tokyo, Japan) utilizing an
XSelect HSS T3 5μm (3.0 x 100 mm) analytical column (Waters Corporation,
Milford, MA). Compounds were separated by gradient elution using 0.1% formic acid
in water and acetonitrile as buffers A and B respectively. A flow rate of 0.5 mL/min
was used throughout. An API-5000 triple quadropole mass spectrometer (ABSciex,
Framingham, MA) utilizing atmospheric pressure chemical ionization (APCI) was
used for analysis. Data was acquired in Multiple Reaction Monitoring mode (MRM)
and analysis time was 5 minutes. Mass transitions for quantification were monitored
for 6-TG (m/z 168/107) and 6-MMP (m/z 167/152). Quantitation was carried out
using the internal standard (IS) ratio method with 8-Bromoadenine. The method was
evaluated in accordance with standard protocols. Method comparison was carried out
by comparing analysis of 70 samples from IBD patients receiving thiopurine therapy
with a reference LC/MS/MS method.
Results: Optimal assay performance was achieved using a minimum volume of 500
μL whole blood. Total assay imprecision was determined for 6-TG at 0.97 (RSD =
13.4%), 3.24 (RSD = 9.3%) and 11.9 (RSD = 9.2%) pmol/0.8 Brbc (where B = 8x10 8 ).
Imprecision was determined for 6-MMP at 10 (RSD = 14%), 41 (RSD = 10%) and
109 (RSD = 12%) pmol/0.8 Brbc. LOQ for 6-TG and 6-MMP were 0.05 μmol/L and
0.5 μmol/L respectively. The correlation between our method and the comparative
method was favorable for 6-TG (R = 0.95; slope = 0.91; intercept = 30.2 pmol/0.8
Brbc) and 6-MMP (R = 0.92; slope = 0.91; intercept = 734 pmol/0.8 Brbc). Values
produced using the reference method were moderately higher for both 6-TG (mean
bias: 7 pmol/0.8 Brbc; 95% limits of agreement: -77 to 90) and 6-MMP (mean bias:
412 pmol/0.8 Brbc; 95% limits of agreement: -2031 to 2854).
Conclusion: We have developed a LC/MS/MS method for measurement of 6-TG
and 6-MMP using minimal sample volume for pediatric applications. Excellent
chromatographic separation was achieved within a run time of 5 minutes using a
modified C18 column exhibiting enhanced retention for the polar thiopurines. Our
MS method utilizes APCI ionization enabling the detection of the molecular ion
species at high abundance. Assay performance was acceptable for imprecision and
bias. Method comparison data from 70 patients undergoing thiopurine therapy showed
favorable correlation with a reference method. The clinical interpretation of data was
also consistent between the two methods.
A-165
Identification of plasma biomarkers of chronic drug exposure in a rat
model
X. Xie 1 , D. Lopez-Ferrer 1 , G. Gil 1 , Y. Karpievitch 1 , B. Nguyen 1 , K. Carr 2 ,
H. Schulman 1 , D. Chelsky 3 , S. Roy 1 . 1 Caprion Proteomics US LLC, Menlo
Park, CA, 2 New York University, New York, NY, 3 Caprion Proteome Inc.,
Montreal, QC, Canada
Background: Identifying patients in need of treatment for substance use disorder
and monitoring their drug use is a necessary step for effective treatment. Previous
proteomic studies of drug abuse or exposure focus on the analysis of cell lines or
tissues as the detection of affected plasma proteins is very challenging. The goal of
this study was to test the feasibility of detecting changes in plasma proteins following
chronic drug exposure in a rat model. The specific aims were to detect and validate
proteomic biosignatures that correlate with behavioral and neurochemical sequelae
in animal models of cocaine, morphine, and nicotine exposure. This initial discovery
strategy could then be used to identify patients predisposed to repeated drug use and
thereby facilitate early intervention.
Methods: Plasma was obtained from rats receiving chronic treatment with cocaine
or with a methylphenidate challenge following end of cocaine administration,
or morphine or nicotine. Depletion of abundant proteins was utilized to yield
low-abundant proteins followed by trypsin digestion and peptide desalting. Peak
alignment, extraction, and label-free quantitation of isotope group components was
conducted followed by intensity normalization. Component differential expression
was obtained by multivariate statistical analysis. After peptide profiling by nano-liquid
chromatography-mass spectrometry (nano-LC-MS) and sequencing by LC-MS/MS,
protein differential expression and identification were achieved. Mass spectrometricbased
multiple reaction monitoring (MRM) assays are being performed in a second set
of animals to verify these potential biomarker candidates.
Results: Only 1 μL of crude rat plasma before depletion was sufficient for downstream
analysis including nano-LC-MS and LC-MS/MS and allowed us to quantify and
identify differentially expressed rat plasma proteins following chronic cocaine,
morphine or nicotine administration as compared to saline. In addition, expression
of plasma proteins evoked by a methylphenidate challenge following end of chronic
cocaine administration were measured. In total, ~500 rat plasma proteins were
identified by LC-MS/MS. Among them, more than 50 proteins were differentially
expressed at 0, 3, 15, and 30 days after termination of 14 consecutive days of chronic
cocaine administration. We found different patterns of changed proteins including
consistent change, early change, or late change in the four time points monitored for
the cocaine cohort compared to saline treated cohort. Some proteins were correlated
with neuron or brain function in response to drug administration. We also measured
the altered expression of plasma proteins following administration of morphine and
nicotine, as well as changes evoked by methylphenidate challenge at 0 and 30 days
following end of chronic cocaine administration. Targeted MRM assays are being
conducted in an independent cohort to validate the protein changes.
CLINICAL CHEMISTRY, Vol. 59, No. 10, Supplement, 2013
A47