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AOAC Official Method 2015.12
Screening and Identification of Phosphodiesterase Type 5 Inhibitors
in Dietary Ingredients and Supplements
Liquid Chromatography/Quadrupole–Orbital Ion Trap Mass Spectrometry
First Action 2015
(Applicable to screening and identification of acetaminotadalafil, acetildenafil, avanafil, homo sildenafil,
hydroxyacetildenafil, hydroxyhomosildenafil, hydroxythiohomosildenafil, lodenafil carbonate,
mirodenafil, propoxyphenyl homohydroxysildenafil, sildenafil, tadalafil, thiohomosildenafil, udenafil,
vardenafil and other known and novel analogues of the above PDE5 inhibitors.)
A.
Apparatus
(a)
LC-MS system.
—UltiMate 3000 LC system (or an equivalent LC system) with Q-Exactive Plus MS
(Thermo Fisher Scientific; San Jose, CA, USA) equipped with electrospray ionization [or an
equivalent high resolution MS/MS) instrument.
(b)
Analytical balances.
—Accurate to two- and four-decimal places.
(c)
Gilson positive displacements pipets.
—Assorted for 100-1000 µL.
(d)
Repeater pipet.
—For 10 µL - 50-mL size tips.
(e)
Horizontal shaker.
—Shaking speed at least 250 rpm.
(f)
Centrifuge.
—Relative centrifugal force (rcf) at least 3000
g
.
(g)
Volumetric flasks.
—Class A, glass, assorted sizes.
(h)
Laboratory glassware.
—Class A, various.
(i)
Disposable polypropylene centrifuge tubes.
—15 and 50 mL.
(j)
Disposable plastic syringes.
—3 mL.
(k)
Syringe filters.
—PVDF, 0.22 µm.
(l)
LC vials and caps.
(m)
Chromatographic column.
—Thermo Fisher Scientific Accucore aQ, 2.6 µm, 100 x 2.1 mm, Part
No. 17326-102130.
B.
Materials and Reagents
(a)
Methanol (MeOH).
—LC-MS and HPLC grade.
(b)
Water (H
2
O).
—LC-MS grade or deionized.
(c)
Acetonitrile (ACN).
—LC-MS and HPLC grade.
(d)
Chloroform.
—HPLC grade.
(e)
Ammonium acetate (NH
4
OAc).
—LC-MS grade.
(f)
Formic acid (FA).
—LC-MS grade.
C.
Reference Standards
Reference standards (purity ≥ 95%) listed in Table
2015.12A
were purchased from Toronto Research
Chemicals (Toronto, ON, Canada), Cachesyn (Mississagua, ON, Canada), TLC Pharmachem (Vaughan, ON,
Canada), and Sigma-Aldrich (St. Louis, MO, USA).
D.
Preparation of Reagent Solutions and Standards
(a)
50:50 (v/v) ACN:H
2
O.
—Combine 500 mL of HPLC grade ACN and 500 mL of deionized H
2
O.
Sonicate for 2 min.
(b)
70:30 (v/v) H
2
O:ACN.
—Combine 700 mL of deionized H
2
O and 300 mL of HPLC grade ACN.
Sonicate for 2 min.
Candidates for 2016 Method of the Year
138