Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 4. Representative chromatograms of MK-2662 in human plasma using LC/LC-MS/MS analysis: (A) single blank from protein purification,
(B) LLOQ at 1 nM from protein purification, (C) single blank from immunoaffinity purification, and (D) LLOQ at 2 nM from immunoaffinity purification
(left panel, MK-2662; right panel, ISTD).
samples to the peak areas of neat standards prepared in the same
solvent and injected directly. The results (Table S2 in the
Supporting Information) show that recoveries ranged from 100.07%
to 119.36%, and matrix effects ranged between 91.32% and 97.2%
across the tested concentrations. On the basis of the intraday
precision results obtained using six different lots of control plasma
(Table 1), an absolute matrix effect should not have any impact
on assay precision.
Validation of the IAP 2D LC-MS/MS Method. The accuracy
and precision of the IAP 2D LC-MS/MS was accessed
and compared with the PPT 2D LC-MS/MS method. The IAP
6884 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
intraday validation data obtained from three standard curves
constructed in three lots of human plasma over the range of 2-200
nM are presented in Table 1, and the QC precision and accuracy
data from three sets of QCs at each of 3, 6, 50, and 150 nM MK-
2662 are shown in Table 2. The chromatograms showed that there
was no interference in the blank and an adequate single-to-noise
ratio for the analyte peak at the LLOQ (Figure 4, parts C and D).
Both methods demonstrated good precision and accuracy, and
met the acceptance criteria for handling small molecules, specified
in FDA guidelines. 34 In comparison to the PPT assay, the IAP
method has a 2-fold higher LLOQ. This was due to differences in