Improved enantioselective assay for the determination of fluoxetine and norfluoxetine enantiomers in human plasma by liquid chromatography

doi:10.1016/S1570-0232(02)00820-6

Journal of Chromatography B

Volume 784, Issue 2, 5 February 2003, Pages 375-383

https://doi.org/10.1016/S1570-0232(02)00820-6 Get rights and content

Abstract

A simple and innovative assay is described which allows the chiral separation of the four enantiomers of fluoxetine and norfluoxetine, with performance characteristics adequate for therapeutic drug monitoring. The assay requires liquid–liquid extraction into acetonitrile/n-hexane/isopropylic alcohol and re-extraction into phosphoric acid for clean-up. The acidic layer is injected onto the HPLC system after filtering. Separation of the analytes is achieved with a Chiralcel ODR column and a mobile phase consisting of potassium hexafluorophosphate/acetonitrile. Detection is made by ultraviolet absorbance at 227 nm. Standard curves are linear for each enantiomer (r²≥0.992) over the range of 10–1000 ng/ml with a limit of quantification of 10 ng/ml for each enantiomer. Within-day and between-day CV% are ≤10% for each enantiomer.

Introduction

Although the selective serotonin reuptake inhibitor fluoxetine has been available for over 15 years and continues to be increasingly used for the treatment of depression and numerous other neuropsychiatric disorders, factors affecting its efficacy and safety have not been fully characterized. Among these, the occurrence of a marked pharmacokinetic variability is likely to play a major role [1], [2], [3], [4]. Assessment of potential correlations between clinical response and plasma levels of fluoxetine and/or its active demethylated metabolite norfluoxetine, however, has been hampered for many years by lack of an adequate assay for the discrimination of the respective enantiomers. Such a discrimination is essential because the chiral forms of fluoxetine and norfluoxetine (Fig. 1) differ considerably in pharmacological activity: in particular, the S-enantiomer of norfluoxetine is 20 times more potent than the R-enantiomer in inhibiting serotonin reuptake both in vitro and in vivo [5], [6], [7], [8].

Many analytical methods based on gas-chromatography (GC, Refs. [9], [10], [11], [12], [13]) and high-performance liquid chromatography (HPLC, Refs. [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]) have been validated for the determination of fluoxetine and its metabolite, but only a few of these allow chiral separation of the enantiomers through derivatization with a chiral agent [10], [11], [13], [28], [29] or use of a chiral stationary phase without derivatization [30], [31], [32], [33], [34]. Moreover, some of the methods using chiral columns only allow separation of the enantiomers of the parent drug (or its metabolite) and cannot be used to determine all four enantiomers in a single run [30], [31].

The goal of the work described in this article was to develop a simpler and faster enantioselective assay for fluoxetine and norfluoxetine, which could be applied for the simultaneous monitoring of the enantiomers of these compounds in the plasma of patients receiving fluoxetine treatment.

Section snippets

Standard, reagents and solvents

Racemic fluoxetine hydrochloride and norfluoxetine hydrochloride were obtained from Eli Lilly (Indianapolis, IN, USA). Pure R- and S-enantiomers of fluoxetine and norfluoxetine were kindly donated by Professor P. Baumann (Prilly-Lausanne, Switzerland). Fluvoxamine maleate (internal standard, I.S.) was obtained from Tocris Cookson Ltd (Avonmouth, Bristol, UK). HPLC grade solvents (acetonitrile, methanol, n-hexane) and phosphoric acid (85%) were purchased from Merck (Darmstadt, Germany); sodium

Chromatographic separation

Under the chromatographic conditions described above, optimal separation of the enantiomers was obtained with peak retention times (RT) of 12.5 min for fluvoxamine (I.S.), 17.5 min for S-norfluoxetine, 18.7 min for R-norfluoxetine, 20.1 min for S-fluoxetine and 21.3 min for R-fluoxetine (Fig. 2). The enantioselectivity factors (α) were 1.10 for fluoxetine and 1.09 for norfluoxetine.

Linearity and sensitivity

The calibration curves for each enantiomer were linear over the concentration range investigated, with mean slopes

Discussion

This method offers significant advantages in terms of simplicity and turnaround time compared with the majority of assays described to date [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]. Time consuming solvent evaporation and sample reconstitution are avoided because the required sensitivity is retained without need for sample concentration steps. Moreover, separation of the R- and S

Acknowledgements

We wish to thank Professor Pierre Baumann (Prilly-Lausanne) for a kind gift of R- and S-enantiomers of fluoxetine and norfluoxetine, and Eli Lilly, Co. (Indianapolis, IN, USA) for supplying a sample of fluoxetine and norfluoxetine racemates. We also wish to thank Dr Anna Bartoli, Dr Alessandro Palmeri and Dr Luigi Fiorina for assisting with the collection and assay of the samples. This study was supported by grants 85689990, 96/H/T14 and 93-99/H/T11 from the Italian National Institute of Health

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Improved enantioselective assay for the determination of fluoxetine and norfluoxetine enantiomers in human plasma by liquid chromatography

Abstract

Introduction

Section snippets

Standard, reagents and solvents

Chromatographic separation

Linearity and sensitivity

Discussion

Acknowledgements

Pharmacol. Ther.

Clin. Ther.

Neuropharmacology

J. Chromatogr. B

J. Chromatogr. B Biomed. Sci. Appl.

J. Chromatogr. B Biomed. Sci. Appl.

J. Chromatogr. B Biomed. Sci. Appl.

J. Chromatogr. B Biomed. Sci. Appl.

J. Pharm. Biomed. Anal.

J. Chromatogr.

J. Chromatogr. A

J. Chromatogr.

J. Chromatogr. B Biomed. Sci. Appl.

J. Pharm. Biomed. Anal.

Br. J. Psychiatry

Ther. Drug Monit.

J. Med. Chem.

Drug Dev. Res.