Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
Analytical Chemistry Chemical Cytometry Quantitates Superoxide
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Figure 1. <strong>Chemical</strong> structures of MK-2662 and its internal standard,<br />
[ 13 C18, 15 N2] MK-2662 (ISTD). The underlined region indicates the<br />
N-terminal tryptic peptide used as surrogate for quantification.<br />
is time-consuming; therefore, reagent availability could become<br />
a rate-limiting step. In the case of MK-2662 analysis, ELISA was<br />
abandoned because a drug-specific antibody was not available at<br />
the time of assay development.<br />
Bioassay is a cell-based or enzyme-based assay that measures<br />
the biologic activity of a specific biological process. 7 Since bioassay<br />
measures the total activity of the sample irrespective of the<br />
chemical structures, the assay specificity is usually less than that<br />
of ELISA. It could provide a measure of pharmacodynamic (PD)<br />
properties 7 but may not be appropriate for pharmacokinetic (PK)<br />
assessment.<br />
Liquid chromatography-mass spectrometry (LC-MS) has<br />
been increasingly used to quantify peptides and proteins in<br />
biological matrixes 9-11 because of its selectivity. Despite commonly<br />
used sample cleanup techniques, such as protein precipitation<br />
(PPT), 9 solid-phase extraction (SPE), 12-14 two-dimensional<br />
(2D) SPE, 15 and 2D high-performance liquid chromatography<br />
(HPLC), 16,17 the immunoaffinity purification (IAP) coupled with<br />
LC-MS/MS represents an emerging strategy for peptide and<br />
protein bioanalysis. The IAP strategies include immunoaffinity<br />
depletion that can be used to remove abundant proteins from<br />
biological matrixes 18,19 and immunoaffinity capture that utilizes<br />
a single antibody to isolate and enrich the target peptides or<br />
(9) van den Broek, I.; Sparidans, R. W.; Schellens, J. H. M.; Beijnen, J. H.<br />
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6878 <strong>Analytical</strong> <strong>Chemistry</strong>, Vol. 82, No. 16, August 15, 2010<br />
proteins from biological samples. Although many applications<br />
using online immunoaffinity columns have been reported, 20-24<br />
offline affinity purification using different carriers, such as<br />
macroporous polymeric beads, agarose or sepharose beads, 25,26<br />
and magnetic beads, 27-29 etc., for immobilization of a variety of<br />
enzyme and/or capture antibodies enables more flexibility in<br />
terms of selection of carriers, antibodies, assay formats, and<br />
experimental conditions as compared to the online approach.<br />
This report presents a sensitive and reproducible bioanalytical<br />
approach for quantification of a PEGylated peptide in plasma. To<br />
support human clinical studies for MK-2662, a 2D HPLC-MS/<br />
MS method using a turbo ion spray (TIS) interface, monitoring<br />
the surrogate N-terminal peptide (HAibDGTFTSDYSK) of MK-<br />
2662 after tryptic digestion, has been developed and validated to<br />
quantify MK-2662 in human plasma. A stable-isotope-labeled<br />
internal standard, [ 13 C18, 15 N2] MK-2662, was employed. Two<br />
sample preparation methods, PPT versus IAP using an anti-<br />
PEG antibody, followed by trypsin digestion in a 96-well format<br />
using 0.2 mL plasma sample, have been evaluated and applied<br />
to clinical sample analysis. The potential caveats of the protein<br />
precipitation approach and the benefit of immunoaffinity<br />
purification in regard to assay specificity are discussed. To our<br />
best knowledge, this is the first report describing use of anti-<br />
PEG antibody to capture a PEGylated peptide in combination<br />
with LC-MS/MS analysis. This approach is potentially applicable<br />
to analysis of other PEGylated peptides or proteins.<br />
EXPERIMENTAL SECTION<br />
<strong>Chemical</strong>s and Reagents. MK-2662 and stable-isotope-labeled<br />
internal standard (ISTD, Figure 1) were synthesized at the Merck<br />
Research Laboratories, Merck & Co. (Rahway, NJ). HPLC grade<br />
acetonitrile, HPLC grade methanol, laboratory grade formic acid<br />
(88%), certified ammonium formate, and certified ammonium<br />
bicarbonate were obtained from Fisher Scientific (Pittsburgh, PA).<br />
Human control plasma (K2EDTA as anticoagulant) was purchased<br />
from Biological Specialty Co. (Colmar, PA). Water was<br />
purified by a Milli-Q ultrapure water system from Millipore<br />
(Bedford, MA). Bovine serum albumin (BSA) at 22% in 0.85%<br />
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Chem. 2008, 80, 1290–1296.<br />
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Jendresen, C. B.; Kimura, M.; Kraft, E.; Lindemose, S.; Lu, J.; McLain, T.;<br />
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Lin, S. H. Protein Expression Purif. 2008, 62, 223–229.<br />
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