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V
aclavik
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
1, 2016
55
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.comDOI: 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.
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
Candidates for 2016 Method of the Year
156