140 / 363
(c)
LC Mobile Phase A.
—Weigh 0.63 ± 0.01 g of NH
4
OAc into appropriate reservoir, add 1000 mL of
H
2
O and 1 mL of FA. Mix thoroughly.
(d)
LC Mobile Phase B.
—Weigh 0.63 ± 0.01 g of NH
4
OAc into appropriate reservoir, add 500 mL of
MeOH and sonicate for approx. 3 minutes. Add 500 mL of ACN and 1 mL of FA. Mix thoroughly.
(e)
Individual Stock Solutions.
—Prepare individual solutions of PDE5 inhibitors at concentrations
ranging from 1500 to 4000 mg/mL. For aminotadalafil, benzyl sildenafil, chloropretadalafil,
desmethylene tadalafil, lodenafil carbonate, tadalafil, and thioaildenafil use a mixture of MeOH
and chloroform (2:1,
v/v
). For the remaining analytes use MeOH. If needed sonicate at approx.
30°C to allow for complete dissolution of the solid standard.
(f)
Mixed Stock Standard Solution.
—Combine individual stock solutions of analytes to prepare a
composite solution at 20 µg/mL in MeOH.
(g)
Internal Standard (ISTD) Solution.
—Prepare a solution at 20 µg/mL in MeOH using a stock
solution of pyrazole N-demethyl sildenafil-
d
3
.
(h)
QC Solvent Standard.
—Accurately transfer 125 µL of the Mixed Stock Standard Solution and 125
µL of the Internal Standard Solution into a 10-mL volumetric flask. Dilute to volume with 70:30
(
v/v
) H
2
O:ACN solution.
E.
Safety
See AOAC Official Methods of Analysis (2005), Appendix B: Laboratory Safety. Use appropriate personal
protective equipment such as lab coat, safety glasses, rubber gloves, and a fume hood. Dispose of
solvents and solutions according to federal, state, and local regulations.
F.
Sample Preparation
(a)
Homogenization and Storage of Samples.
—Solid samples such as botanical powders, extracts
and tablets are blended to obtain homogenous samples and stored at –4°C. Softgels, gelcaps
and capsules are homogenized using cryogenic grinding with liquid nitrogen and stored at –
70°C. Liquid samples are briefly shaken and stored at –4°C.
(b)
Extraction Procedure.
1. Weigh 1.00 ± 0.02 g of thoroughly homogenized sample into a 50-mL centrifuge tube.
2. Add 20 mL of 50:50 (
v/v
) ACN:H
2
O solution, briefly hand-shake / vortex and then shake for
15 minutes using a horizontal shaker set at approximately 250 rpm.
3. Centrifuge the tube at >3000
g
for 5 minutes.
4. Transfer 1 mL of the supernatant to another 50-mL centrifuge tube.
Note: When transferring extract aliquots obtained for softgels, avoid the upper lipophilic
layer formed during the centrifugation step.
5. Add 19 mL of 70:30 (
v/v
) H
2
O:ACN solution and vortex mix briefly.
6. Filter approximately 3-mL of the diluted extract using plastic syringe fitted with a 0.22 µm
PVDF syringe filter into a 15-mL centrifuge tube.
7. Transfer 1 mL of the filtrate to a 2-mL autosampler vial and add 12.5 µL of the Internal
Standard Solution.
8. Cap the vial and vortex mix briefly.
9. Perform LC-HRMS analysis.
G.
LC-HRMS Analysis
(a)
LC Operating Conditions.
1. Column:
Thermo Scientific Accucore aQ, 2.6 µm, 100 x 2.1 mm
Candidates for 2016 Method of the Year
139