Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 3. Representative chromatograms of the N1-12-mer [m/z 672 f 223] from MK-2662 plasma sample analyzed using (A) 1D LC-MS/MS
or (B) 2D LC-MS/MS. The left panel of part B is the chromatogram from the SCX column (the first dimension), where T18-30 and T19-30 were
the peptides obtained from trypsin digestion of MK-2662. The right panel of part B is from the RP column (the second dimension). The total run
time for 2D LC-MS/MS was 4.5 min per sample.
problem for preparing control plasma in the analysis laboratories.
Therefore, a test was conducted to determine equivalence of BD-
P700 inhibitor tubes to Linco brand DPP-IV inhibitor that was
available in easy to use solution form. The results demonstrated
that the stabilizers from BD tubes (1.5 mL of plasma per tube)
and Linco inhibitor (20 µL/mL plasma) provided comparable
effects in terms of stability (including freeze-thaw, benchtop,
autosampler, and reinjection stabilities), matrix effect, and recovery
in human plasma.
Optimization of Trypsin Digestion Conditions. A surrogate
proteolytic peptide approach using trypsin digestion was selected
for the MS/MS-based quantification of MK-2662 in human plasma.
Following protein precipitation, the supernatant was brought to
dryness and then reconstituted in NH 4HCO3 buffer that contained
trypsin for digestion. This step needed to be optimized to
ensure rapid, complete, and reproducible digest within a
reasonable time frame.
The digestion yield was tested by varying the amount of
trypsin from 2 to 10 µg, the volume of plasma in the BD-P700
tube, and different digestion time at 2, 3, 4, and 16 h in both
Linco DPP-IV-containing plasma and the BD-P700-treated
plasma, where the latter appeared to need more trypsin due to
the inhibitory effect of proprietary enzyme inhibitors. The
reason for testing plasma volume was that, at the clinical site,
although 3 mL of blood per sample was required in BD tube,
there was a chance that less volume was collected. Since the
BD tubes contain a fixed amount of protease inhibitor, the lower
the blood volume, the higher the inhibitor concentration in the
tube, which may lead to the more difficult trypsin digestion.
6882 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
The tested plasma volume at 1 and 1.5 mL covered a range of
2-3 mL of blood collection.
The test results are shown in Figure S2 (see the Supporting
Information), where the experiment was conducted at 10 nM MK-
2662 in all samples, and the yield was calculated assuming 100%
digestion in a neat MK-2662 solution incubated with trypsin for
3 h. The spiked plasma without inhibitor appeared to give higher
yield compared to the neat sample, presumably due to surface
absorption loss of the neat sample. The data indicated that a
digestion with 7 µg of trypsin was very sensitive to the change of
blood volumes1 mL of plasma (equivalent to 2 mL of blood
collection) required much longer digestion time to achieve >80%
digestion. In contrast, a digestion with 10 µg of trypsin demonstrated
an improved tolerability, yielding nearly complete digestion
within 3-4 h for both 1 and 1.5 mL of plasma (equivalent to 2-3
mL of blood collected in the BD-P700 tube). Balancing the benefit
and risk by considering digestion yield and sample processing
time, the digestion conditions10 µg of trypsin for 3 hswas
selected. The digestion outcome under these conditions was
robust and highly reproducible. Consequently, the clinical site was
instructed to collect 3 mL of blood (1.5 mL plasma equivalent)
and to record significant deviation from this volume.
Solvent for Working Standard Solutions. It is well-known
that peptides are prone to absorption loss to container surfaces. 9
Initially, 30% acetonitrile was used for preparation of both primary
stock and working standard solutions. Following storage at 4 °C
for 3 weeks, the primary stock was stable; however, more than
30% of MK-2662 loss was observed in working stock solutions.
Switching the working stock solvent to 1% BSA and storing at