5. AOACSPDSMethods-2018AwardsV3

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V aclavik et al .: J ournal of AOAC I nternational V ol . 99, N o . 1, 2016  67

Figure 2015.12. Detection/identification workflow for targeted analytes.

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

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