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