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Orthogonal Assay for Fluoxetine
Simple methods without the use of ion-pairing agents
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printable Application Sheet
Notes:
Fluoxetine is a widely prescribed antidepressant which acts by selective inhibition of presynaptic serotonin reuptake. In addition, fluoxetine
can also act as a noncompetitive antagonist of nicotinic acetylcholine receptors. Sold as a racemic mixture, fluoxetine’s R and S
forms show similar efficacy in vivo, and its binding affinity has been shown to be largely stereoindependent.
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Method Conditions
| Column |
Fig. A: Phenyl Hydride™, 4µm, 100A
Fig. B: Diamond Hydride™, 4µm, 100A |
| Catalog No. |
Fig. A: 69020-7.5P Fig. B: 70000-7.5P |
| Dimensions |
4.6 x 75 mm |
| Solvents |
Fig. A: DI water/ 0.1% formic acid
& nbsp; Fig. B - 97% Acetonitrile/ 3% DI water/ 0.1% formic acid |
| Gradient |
| Fig. A |
Fig. B |
| time (min.) |
%B |
time (min.) |
%B |
| 0 |
10 |
0 |
95 |
| 6 |
90 |
6 |
60 |
| 7 |
10 |
7 |
95 |
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| Flow rate |
1.0 mL/min |
| Detection |
UV 228 nm |
| Injection Volume |
10µL |
| Temperature |
Fig. A: 35° C Fig. B: ambient25° C |
| Sample |
Fluoxetine capsule extract
Stock Solution: 20 mg strength capsule contents added to 100 mL volumetric flask, diluted to mark with 50:50 A:B, Vortexed 5 min,
sonicated 5 min, then filtered through a 0.45 µm nylon membrane (MicroSolv Technology Corp. Eatontown, NJ, USA). Working Solution: 100
µL stock diluted with 900 µL 50:50 A:B |
| Peak |
1. Fluoxetine |
Discussion
The USP assay methods for fluoxetine use ion-pairing agents in the mobile phase in order to reduce analyte peak tailing due to silanolic interactions.
However, this peak tailing is not an issue with this method since these silanols are mostly replaced with Si–H groups. A disadvantage of ion-pairing
agents is their slow uptake and release from the HPLC column, resulting in long equilibration times and poor repeatability. Both figures show how
excellent peak symmetry can be achieved either in the reversed phase (RP, Figure A) or aqueous normal phase (ANP, Figure B) mode with only formic acid
as the mobile phase additive. The ability to perform the assay in either the RP or ANP mode is highly beneficial for development of orthogonal
analytical methods. Chromatograms show 5 runs overlayed to highlight precision.
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