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V
aclavik
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
1, 2016
67
mixture containing equal amounts of MeOH and ACN was used
as the organic component of the mobile phase (
see
Figure 1).
Under optimized conditions, analytes eluted between 3 and
15 min of the run with typical at-base peak widths ranging from
12 to 18 s. Of eight isobaric analyte groups, each containing two
to four compounds, all analytes could be chromatographically
resolved.
MS/MS Spectra
The availability of MS/MS data are crucial for reliable
screening and identification of both known PDE5 inhibitors
and their novel analogs. The MS/MS spectra of analytes were
recorded in data-dependent product ion scan mode through the
isolation and fragmentation of their respective pseudomolecular
ions and in AIF mode. Rather than performing fragmentation
at a single NCE setting, three discrete values of 40, 70, and
100% were used. This stepped NCE approach allowed obtaining
fragments stable under different collision energies in a single
MS experiment and resulted in information-richMS/MS spectra.
Based on the review of the MS/MS spectra of all analytes,
product ions frequently occurring in records of parent PDE5
inhibitors and their analogs were found. For example, fragment
ion exact masses
m/z
377.12780, 311.15025, 299.09611,
285.13460, 283.11895, and 99.09167 were frequently
present in fragmentation spectra of sildenafil and its analogs,
fragment
m/z
204.08078 was characteristic of tadalafil and
its analogs, and fragment ions
m/z
123.09167 and 110.06004
were characteristic of vardenafil and its analogs. A combined
Figure 2015.12. Detection/identification workflow for targeted analytes.
43