5. AOACSPDSMethods-2018AwardsV3

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V aclavik et al .: J ournal of AOAC I nternational V ol . 99, N o . 1, 2016  55

Single-Laboratory Validation Study of a Method for Screening and Identification of Phosphodiesterase Type 5 Inhibitors in Dietary Ingredients and Supplements Using Liquid Chromatography/Quadrupole–Orbital Ion Trap Mass Spectrometry: First Action 2015.12 L ukas V aclavik Covance Laboratories, Otley Rd, Harrogate, United Kingdom, HG3 1PY J ohn R. S chmitz Covance Laboratories, 3301 Kinsman Blvd, Madison, WI 57304 J ean -F rancois H albardier Covance Laboratories, Otley Rd, Harrogate, United Kingdom, HG3 1PY K aterina M astovska 1 Covance Laboratories, 3301 Kinsman Blvd, Madison, WI 57304 Dietary Supplement

Received August 27, 2015. This method was approved by the Expert Review Panel for Dietary Supplements as First Action. The Expert Review Panel for Dietary Supplements invites method users to provide feedback on the First Action methods. Feedback from method users will help verify that the methods are fit-for-purpose and are critical for gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author or methodfeedback@aoac.org. 1 Corresponding author’s e-mail: katerina.mastovska@covance.com DOI: 10.5740/jaoacint.15-0202 A single-laboratory validation study of a method for screening and identification of phosphodiesterase type 5 (PDE5) inhibitors in dietary ingredients and supplements is described. PDE5 inhibitors were extracted from the samples using a 50:50 (v/v) mixture of acetonitrile and water and centrifuged. Supernatant was diluted, filtered, and analyzed by LC–high-resolution MS. Data were collected in MS acquisition mode that combined full-scan MS experiment with all-ion fragmentation and data-dependent MS/MS product from the ion scan experiment. This approach enabled collection of MS and tandem MS (MS/MS) data for both targeted and nontargeted PDE5 inhibitors in a single chromatographic run. Software-facilitated identification of targeted analytes was performed based on the retention time, accurate mass, and isotopic pattern of pseudomolecular ions, and accurate masses of fragment ions using an in-house compound database. Detection and identification of other PDE5 inhibitors and novel analogs were performed by retrospective evaluation of MS and MS/MS experimental data. The method validation results obtained for evaluated matrixes fulfilled the probability of identification requirements and probability

of detection requirements (for the pooled data) set at 90% (95% confidence interval) in the respective AOAC Standard Method Performance Requirements for identification and screening methods for PDE5 inhibitors. Limited data demonstrating the quantification capability of the method were also generated. Mean recovery and repeatability obtained for the evaluated PDE5 inhibitors were in the range 69–90% and 0.4–1.8%, respectively. D eliberate addition of active pharmaceutical ingredients to dietary supplements is a profit-driven practice that aims to develop or intensify the claimed biological effect of the product (1, 2). Phosphodiesterase type 5 (PDE5) inhibitors, such as avanafil, lodenafil carbonate, mirodenafil, sildenafil, tadalafil, udenafil, or vardenafil and their unapproved designer analogs, represent an important class of pharmaceuticals that are frequently used to adulterate products advertised to provide an enhancement to sexual performance and ingredients used in their manufacturing (3, 4). Considering that PDE5 inhibitors can negatively interact with certain prescription drugs and that limited knowledge is available on safety and efficacy of the designer analogs, the presence of such compounds in dietary supplements may represent a serious health risk to consumers (2). Therefore, reliable analytical methods are needed for detection, identification, and quantification of PDE5 inhibitors in relevant dietary supplement raw materials and finished products. To address this problem, AOAC INTERNATIONAL issued a call for methods for screening, identification, and determination of PDE5 inhibitors in dietary ingredients and supplements based on Standard Method Performance Requirements (SMPRs ® ) developed by a working group of the AOAC INTERNATIONAL Stakeholder Panel on Dietary Supplements (5–7). Single-laboratory validation (SLV) requirements provided in AOAC SMPR 2014.010 for identification of PDE5 inhibitors are summarized in Table 1.