Development of a stir bar sorptive extraction based HPLC-FLD method for the quantification of serotonin reuptake inhibitors in plasma, urine and brain tissue samples

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Abstract

The aim of this article is to present an analytical application of stir bar sorptive extraction (SBSE) coupled to HPLC-fluorescence detection (FLD) for the quantification of fluoxetine (FLX), citalopram (CIT) and venlafaxine (VLF) and their active metabolitesnorfluoxetine (NFLX), desmethyl- (DCIT) and didesmethylcitalopram (DDCIT) and o-desmethylvenlafaxine (ODV) – in plasma, urine and brain tissue samples. All the parameters influencing adsorption (pH, ion strength, organic modifier addition, volume, extraction time and temperature) and desorption (desorption solvent composition, time, temperature and desorption mode) of the analytes on the stir bar have been optimized. For each matrix, the analytical method has been assessed by studying the linearity and the intra- and interday accuracy (89–113%) and precision (RSD < 13%). The improvement of the quantification limits (0.2–2 μg l−1 for plasma, 2–20 ng g−1 for brain tissue and 1–10 μg l−1 for urine, depending on the respective response for analytes) and the development of a procedure for all the matrices make this method useful in clinical and forensic analysis.

Introduction

Depression, a very frequent psychiatric illness, has become one of the main diseases to be studied by heath institutions. According to WHO (World Health Organization) depression was the fourth leading contributor to the global burden of disease in 2000 and it is expected to be the second in 2020 [1].

Fluoxetine (FLX) and citalopram (CIT) are important SSRI antidepressants usually used in psychiatry. Despite the SSRIs activity, some second generation antidepressant drugs also have a norepinephrine reuptake inhibition activity. These two activities make venlafaxine (VLF) an alternative for patients whose response to SSRIs has decreased. However, VLF can still cause several side effects such as nausea, somnolence, asthenia and headache [2]. FLX, CIT and VLF share the property that some of their metabolites, norfluoxetine (NFLX), desmethyl- (DCIT) and didesmethylcitalopram (DDCIT) and o-desmethylvenlafaxine (ODV), are also pharmacologically active [3] (Fig. 1).

In clinical practice, the determination of an individual optimum dose for an antidepressant drug is often based on a trial-and-error strategy. In the case of antidepressant drugs, therapeutic drug monitoring is a well-established tool for defining a more efficient and safe dose. Furthermore, in some cases the monitorization of the pharmacologically active metabolites could be interesting for the correct dose establishment.

To date, the analytical methods described in the literature for the analysis of non-tricyclic antidepressants in biological matrices usually use liquid–liquid extraction (LLE) [4], [5], [6], [7] or solid-phase extraction [8], [9], [10], [11], [12], [13] for sample preparation. In general, these procedures are laborious, time-consuming and involve multiple steps.

In the recent years, different sorptive extraction techniques have been successfully applied to analyze drugs in biological fluids. Solid-phase microextraction (SPME), where the sorbent coating is applied over a thin silica fiber mounted on a syringe needle, has been proven to be an interesting alternative for the extraction of SSRIs in urine coupled to both gas-chromatography (GC) [14] and liquid-chromatography (LC) [15].

Based on the same principles of those of SPME, stir bar sorptive extraction (SBSE) has been employed as a new sample pretreatment technique. In SBSE, a stir bar is coated with a polydimethylsiloxane (PDMS) layer (0.5–1 mm thick) and used to stir samples, thereby extracting and enriching solutes into the PDMS layer. After extraction, the stir bar is removed and dried with a soft tissue and then, the analytes can be desorbed with thermal desorption for GC analysis. In contrast to SPME, the thermal desorption cannot be done directly in the injection port of a GC equipment and a special interface is required. As an alternative, liquid desorption can also be used coupled to LC, which is an advantage in the determination of thermolabile solutes or those with a low volatility, as well as SSRIs, that require previous derivatization in GC. Although magnetic stirring and sonication have been used, Lambert et al. observed some degradation of the stir bar coating after 30 desorptions when sonication is used [11].

The main differences between SPME and SBSE are the design of the extraction system and the much larger volume of sorbent used in the latter, increased by a factor ranging from 50 to 250 which results in higher sensitivity thus decreasing the detection limits [16].

In biomedical and life science applications, SBSE coupled to GC has been widely used to characterize chemical compounds in urine, plasma, saliva and some gland excretions, while there are fewer methods coupling SBSE to liquid-chromatography [16], [17].

With regard to the extraction of antidepressants with SBSE, most of the published studies are based on the quantification of some of these compounds in plasma samples [18], [19], [20]. However, none of these studies demonstrate the applicability of the developed methods to urine and brain samples.

The aim of this work was to develop an SBSE procedure for the quantification of SSRI antidepressants and their active metabolites in plasma, brain tissue and urine samples. The proposed method should be useful at clinical levels and suitable for studies where the objective was the establishment of a more efficient and safe dose or for screening in clinical samples and in forensic analysis.

Section snippets

Chemical and solutions

All reagents were analytical grade of the highest purity available. Fluoxetine hydrochloride, and venlafaxine hydrochloride were purchased from Sigma (St. Louis, USA). Norfluoxetine oxalate and o-desmethylvenlafaxine were supplied by Cerilliant (Texas, USA). Citalopram, demethylcitalopram and didemethylcitalopram were kindly donated by Lundbeck A/S (Copenhagen, Denmark). The ion-pair reagent tetramethylamonium chloride (TMACl) used in the mobile phase was purchased from Merck (Darmstadt,

Results and discussion

As previously stated, several parameters affecting desorption and extraction steps were evaluated. The first step was the evaluation of desorption parameters such as solvent, time and temperature (Fig. 2). The desorption solvents evaluated were acetonitrile, methanol and mobile phase varying the volume and desorption time from 5 to 30 min. The assay was performed at 20 °C, 50 °C and 75 °C for magnetic stirring and at room temperature for sonication. It was found that the best recovery values were

Conclusions

The developed SBSE/HPLC-FLD procedure permits the quantification of FLX, CIT and NFLX and their active metabolites ODV, DCIT, DDCIT and NFLX minimizing laborious and complicated sample preparation procedures. No additional clean up step is necessary between two SBSE extractions to assure efficient removal of interferences or analytes. Moreover, the use of HPLC makes a previous derivatization step unnecessary. The selectivity of the SBSE procedure together with the selectivity of the

Acknowledgements

This work was supported by the Spanish Minister of Science and Innovation (CTQ2008-00651/BQU). We wish to thank the Central Service of Analysis of the University of the Basque Country for its excellent technical assistance.

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