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Detection of fluoxetine in brain, blood, liver and hair of rats using gas chromatography-mass spectrometry

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Detection of fluoxetine in brain, blood, liver and hair of rats using gas chromatography-mass spectrometry
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  Life Sciences, Vol. 64, No. 9, pp. 805411, 1999 cnpyright 0 1999 elscvier science Inc. Printed in the USA. All right6 rcSeIvcd 0024-3205/99/ -w hoot matter ELSEVIER PII s00243205 98)00622-5 DETECTION OF FLUOXETINE IN BRAIN, BLOOD, LIVER AND HAIR OF RATS USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY Michel Lefehvrel, Mario Marchandl, Judith M. Horowitz2 and German Torres2* %entre de Toxicologic du QuebBc, 2705 Boul. Laurier, Ste-Foy, QC, Canada GlV 4G2 Behavioral Neuroscience Program, Department of Psychology State University of New York at Buffalo, Buffalo, New York 14260 USA (Received in final form November 21, 998 Summary This study reports the measurements of fluoxetine in discrete brain regions, blood, liver and hair of male rats injected with 10 mg/kg fluoxetine HCl for 15 consecutive days. Concentrations of the antidepressant were obtained by gas chromatography-mass spectrometry (GC-MS) methodology. In brain, fluoxetine levels were unevenly distributed, with the raphe nucleus containing the highest amounts relative to the hypothalamus or striatum. Fluoxetine was also measured in blood and liver roughly paralleling those ratios described in previous rodent studies. Of potential interest, fluoxetine waa found to accumulate in rat hair after chronic treatment. Detection of fluoxetine in hair by GC-MS could be used as a marker for probative analyses. Key Words: serotonin, antidepressant, triatum, hypothalamus, aphe nucleus, hair Fluoxetine is a serotonin (5-hydroxytryptamine; 5-HT) re-uptake inhibitor widely used in the pharmacotherapy of endogenous depression. It targets membrane transporters in the central nervous system (CNS) thereby increasing synaptic concentrations of 5-HT molecules. The effectiveness of fluoxetine in potentiating 5-HT neurotransmission appears to be the key process in ameliorating some of the clinical attributes of depression (1,Z). Fluoxetine is a racemic drug mixture containing both R and S enantiomers that readily penetrates the blood-brain barrier (2). In rats, fluoxetine at doses ranging from 5 to 10 mg/kg also increases synaptic concentrations of 5-HT in telencephalic (e.g., striatum), diencephalic (e.g., hypothalamus) and mesencephalic (e.g., raphe nucleus) subdivisions of the brain parenchyma (3-5). Although the neurochemical consequences of 5-HT re-uptake inhibition by fluoxetine are well delineated, very little is known about the relative concentrations of the aforementioned antidepressant in rat brain, particularly after chronic administration. In rats and humans, the half-life (tI/2) of fluoxetine is approximately l-2 days (6). The slow elimination of fluoxetine may be of importance in relation to its pharmacokinetic profile at brain sites following systemic administration. Therefore, using gas chromatography and mass-spectrometrv (GC-MS) methodology, we measured the relative levels of fluoxetine in *Corresponding Author: Dr. German Torres, Behavioral Neuroscience Program, Department of Psychology, Park Hall, State University of New York at Buffalo, Buffalo, New York, 14260 USA, Telephone: (716) 645-3650, FAX: (716) 645-3801, e-mail: gtorres@acsu.buffalo.edu  8 6 Fluoxetine Levels n Rats Vol. 64 No. 9 1999 the striatum, hypothalamus and raphe nucleus. The reasons for selecting these brain regions for fluoxetine measurements were based on the fact that both striatal and hypothalamic neurons receive dense axonal 5-.HT input from the raphe nucleus (7), and also because these brain regions appear to play key roles in the pathogenesis of depression. For instance, it is thought that 5-HT-containing nerve cells acting on discrete hypothalamic nuclei that synthesize the hormone-peptide corticotropin-releasing factor (CRF) may be normalized during successful antidepressant pharmacotherapy (8-10). We also measured levels of fluoxetine in blood and liver in order to relate the in uiuo concentrations of this drug to actual brain concentrations of the antidepressant after 15 days of intraperitoneal (IP) administration (10 mg/kg dose) to rats. Finally, we were interested in measuring the relative levels of fluoxetine in terminal hair of rats. Because hair analysis is a noninvasive procedure, it could be used as a comprehensive means of fluoxetine detection over a period of days or months of antidepressant treatment. Additionally, a variety of drugs appear to remain sequestered in the hair shaft over long periods of time (e.g., months to years), thereby providing a wider window of detection not readily available in blood fluoxetine levels as these analytes decrease rapidly over a relatively short period of time (e.g., hours to days). Methods and Materials Animals. Male Long-Evans rats (Holtzman, MN; 250-260 g) were used for all experiments described herein. The animals were housed in groups of two in a vivarium with constant ambient temperature (22 “C), humidity (60 ) and maintained on a 12:12 h 1ight:dark cycle (lights on at 0700 h). All rats had free access to food and water and were handled for a few days prior to the fluoxetine injections in order to minimize the risk of non-specific stress. All testing (see below) was carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and with approval from the State University of New York at Buffalo IACUC. Experimental Procedures. On the first day of fluoxetine treatment, the animals were housed individually and injected IP with fluoxetine hydrochloride (HCl) at a dose of 10 mg/kg (calculated as the free base and dissolved in distilled water). This dose was chosen because it alters the responsivity of rat forebrain neurons to 5-HT (ll), and also because this dose up-regulates ~-HT~A/~C receptor subtypes in limbic structures (12). The rats were injected daily for 15 consecutive days with fluoxetine approximately 1 h before lights off. To verify the proper administration of the antidepressant, treated rats were observed for decreased behavioral activity and body flattening for 10 min after fluoxetine administration (13). Control-rats (n = 3) were injected with distilled water at a dose of I ml/kg. On day 5 of the experiment, fluoxetine- and water-treated rats were gently removed from their respective home-cages and immediately shaven at the level of the neck (just below the ears) with an electric shaver. Special care was taken to clean the shaver thoroughly between animals in order to prevent cross-contamination of hair samples. Additionally, gloves were worn by the investigators to further prevent cross-contamination. Neck hair clippings, approximately 1000-1500 strands varying in lengths from 0.5 to 1 cm, were collected from each rat and placed in sterile glass vials and stored immediately at -20 “C until extraction procedures (see below). On day 15 of the experiment, all rats were shaved again on the same (neck) area and injected for the last time with 10 mg/kg fluoxetine HCl. Thirty min later, all rats were killed by decapitation. Trunk blood was collected in ice-cold polypropylene tubes containing 10 ~1 EDTA (60 mg/ml), and immediately centrifuged at 1500 r.p.m for 10 min. The plasma was divided into aliquots and stored frozen until determination of fluoxetine content.  Vol. 64, No. 9, 1999 Fluoxetine Levels in Rats 807 Liver samples were collected from the anterior right lobe by excising a discrete region of the liver (-20 mg/sample) and placing it directly into ice-cold microtubes containing 1 ml of high grade ‘HPLC water. Each individual liver sample was homogenized with a pestle, centrifuged at 25000 r.p.m for 20 min and the supernatant stored frozen. Brains were rapidly removed from the calvaria and dissected into the striatum (caudate putamen and nucleus accumbens), hypothalamus and raphb nucleus. The extent of the microdissected brain tissue ranged from bregma 2.70 mm (plate 9) to bregma -0.40 mm (plate 20) for the striatum; bregma 1.30 mm (plate 23) to bregma -2.12 mm (plate 26) for the hypothalamus; and bregma -7.04 mm (plate 46) to bregma -8.80 mm (plate 53) for the raphe nucleus. The plates of brain sections chosen were from the Rat Atlas of Paxinos and Watson (14). The mean + SEM amount of tissue (in mg) for the striatum was: 417.5 + 21.5, for the hypothalamus: 30.06 + 1.84 whereas for the raphk nucleus: 130.5 + 13.1. All the aforementioned brain regions were homogenized with a pestle in 1 ml of high grade HPLC water and stored frozen at -70 “C. Differences in brain, blood, liver and hair fluoxetine levels were evaluated by unpaired t-tests with statistically significant differences defined as P 5 0.05. Reagents. The following solvents were used: hexane diethyl-ether and benzene, both HPLC grade, obtained from Fisher Scientific (Sprintield, NJ). Pentafluoropropionic anhydride (PFPA), a derivatization agent, was purchased from Pierce (Rockford, IL). Fluoxetine HCl was kindly donated by Eli Lilly Laboratories (Indianapolis, IN). Digestion Procedures. Aliquots of brain, liver and hair were weighed in glass tubes. A solution consisting of 3 ml of 0.6N NaOH along with 100 ~1 of maprotiline (an internal standard solution) was added to each rat sample and placed for 2 hr in a water bath at 56 “C. Subsequently, the samples were allowed to cool at room temperature and were centrifuged at 1500 r.p.m for 15 min; the supernatant was then transferred to clean 25 ml glass tubes. Extraction Procedures. The digested supernatant samples were coated with 500 pl of a 2N NaOH solution and then extracted with 11 ml of a 1:l hexane:diethyl-ether solution. After vigorous shaking and centrifugation, the top organic layer of each extracted sample was recovered and back-extracted using 5 ml of 0.2 M HCl. A further extraction procedure was performed by using 3 ml of carbonate buffer (pH: 11.7) and 6 ml of a 1:l hexane:diethyl- ether solution. This organic layer was evaporated to dryness and redissolved in 200 ~1 of benzene prior to derivatization. To each extracted sample, 100 1.11 f PFPA was added and the samples were placed in a water bath at 56 “C for 10 min. The samples were then cooled at room temperature, diluted with 5 ml of hexane and their content evaporated to dryness. The derivitized products were then recovered in 500 ~1 hexane. Gas Chromatography and Mass Spectrometry Procedures. The analytical instrumentation consisted of a Varian Saturn II Ion-trap GC-MS apparatus. The gas chromatography (Varian 3500) was equipped with an on-column injection and a 30 m, 0.25 mm id, DB-5 column. The detector was heated to 240 “C and operated in an electron impact ionization mode with an emission current of 23 pamperes. Recovered derivatives in hexane (2 ~1) were injected in the GC. Baseline separation as well as full scan GC-MS identification of peaks were obtained from all samples. Specific sums of (hydrogen) ions were computed for quantification purposes where fluoxetine-pentafluoropropionyl molecules were measured following the exposure of the samples to PFPA. For fluoxetine these were: 117, 190 and 294 amu. Corresponding peak areas counted for a standard of 620 ng 5uoxetine were 300,000 at 10 min. Solutions of 5uoxetine were dissolved in ethanol at a concentration of 4 mmol/l. Successive dilutions in water were instituted at 50 lmol/l, 10 kmol/l and 2 pmol/l. Maprotiline was used as the internal standard at a concentration of 80 pmolil of water.  Boa Fluoxetine Levels in Rats Vol. 64, No. 9, 1999 Standards were carried over all digestive and extraction procedures and the calibration curves that followed were linear between 0.1 and 2.0 pmolfl (60 and 1240 ng for a 1 ml spike). Tests were performed to ensure that the digestion procedure did not compromise the recovery of fluoxetine. In this context, both fluoxetine and maprotiline were stable under the stated digestive conditions. Based upon these procedures, the sensitivity for fluoxetine was 0.25 ng/mg. The index of reproducibility (inter-assay coefficient of variation) was 7.9 , whereas the index of precision (intra-assay coefficient of variation) was 10 . Results Systemic injections of fluoxetine HCl to male Long-Evans rats produced episodes of behavioral inactivity. By and large, all treated-animals showed brief bouts of immobilization and flattening of the body for about 10 min after fluoxetine administration. This behavioral profile was maintained steadily throughout the 15 days of testing (data not shown). Thirty min after the last antidepressant injection, there were considerable (but variable) levels of fluoxetine in the striatum (ranging from 0.01 to 4.7 ng/mg), hypothalamus (ranging from 0.32 to 1.1 ng/mg) and raphe nucleus (ranging from 0.02 to 8.4 ng/mg). The raphk nucleus was the brain region with the highest fluoxetine content, whereas the hypothalamus showed the lowest (Table 1). It should be noted that (ng/mg) levels of the dealkylated derivative metabolite, norfluoxetine, were also detected in brain and paralleled those of the parent drug. For instance, norfluoxetine levels (means + SEM) in the striatum (n = 3) were: 1.77 + 0.32; in the hypothalamus (n = 3): 0.65 + 0.04, and in the raphh nucleus (n = 3): 2.1 + 0.25. As expected, no fluoxetine was detected in comparable brain regions of rats injected with the water-vehicle (data not shown). In general, our results show that fluoxetine (and norfluoxetine) concentrations vary in different brain regions. Indeed, this differential concentration of fluoxetine in rat brain is similar to that reported with other classes of antidepressants, particularly the tricyclic chlorimipramine (15). Fluoxetine content was also detected by CC-MS methodology in blood (ranging from 0.56 to 1.22 mgll) and liver (ranging from 0.04 to 3.58 ng/mg) of rats injected (IP) for 15 consecutive days with 10 mg/kg of the antidepressant. Norfluoxetine levels were detected in blood with (mg/L) concentrations (means + SEM, 0.91 + 0.02; n = 3) roughly paralleling those of fluoxetine. Liver tissue contained the highest content of fluoxetine (Table 2). It should be noted that plasma fluoxetine concentrations were significantly lower than those reported in the striatum and raphe nucleus. Similar ratios are observed in animals treated with other antidepressant drugs (15). Interestingly, on days 5 and 15 of fluoxetine treatment, considerable amounts of the antidepressant were also measured in extracts from rat hair (Table 2). Fluoxetine content in hair was significantly higher (P ( 0.05) on TABLE 1 Gas Chromatography-Mass Spectrometry Measures of Brain Fluoxetine Concentrations1 Brain Region Mean + SEM n Striatum 2.25 + 0.58 10 Hypothalamus 0.68 + 0.11 7 Raphe Nucleus 3.86 & 0.96 10 IBrain (pooled) samples were collected from fluoxetine-treated rats 30 min after the last IP injection(l0 mglkg). Mean + SEM values are ng/mg wet weight.  Vol. 64, No. 9, 1999 Fluoxetine Levels n Rats 809 day 5 than when compared with drug content on day 15. In contrast, terminal hair extracted from rats injected with the water-vehicle on days 5 and 15 was devoid of fluoxetine analytes (data not shown). Therefore GC-MS methodology appears to be a viable approach for the detection of antidepressant concentrations in hair, which may prove to be an excellent marker for probative analyses. Discussion The present study demonstrates that chronic n u uo exposure to fluoxetine results in the variable accumulation of this antidepressant in brain, blood, liver and hair. In selected brain regions, we found that the raphe nucleus exhibited the highest concentration of the drug suggesting therefore that fluoxetine influences 5-HT neurotransmission differently in the brain parenchyma. Indeed, distribution of both 5-HT uptake and transporter sites varies considerably in brain regions, with the raphk nucleus containing significantly greater amounts of the biogenic transporter than other nuclei (16,17). In addition, animal studies show tricyclic concentrations varying in different brain regions (15). It should be noted, however, that the striatum, hypothalamus and raphk nucleus all showed concentrations of fluoxetine that were lo-20 times those blocking 5-HT re-uptake in uitro (2). Therefore, in terms of maximum concentrations (Cmax) and area under the concentration-time curve (AUC), it appears that fluoxetine content in each selected brain region is more than sufficient to saturate 5-HT re-uptake mechanisms. It should also be noted that there was significant intra-animal variability in brain fluoxetine content. It is conceivable that such a variability may be a reflection of individual pharmacogenetic differences. In general, our brain concentrations of fluoxetine roughly paralleled those reported in laminar cortical and hippocampal regions of rats injected acutely with this antidepressant (18). TABLE 2. Gas Chromatography-Mass Spectrometry Measures of Blood, Liver and Hair Fluoxetine Concentrations l Region Mean + SEM n 0.99 + 0.14 5 Liver 2.06 + 0.5 8 Hair (day 5) 9.96 + 1.72* 4 Hair (day 15) 1.12 + 0.26 6 IBlood, liver and hair samples were collected from fluoxetine-treated rats 30 min after the last IP injection (10 mg/kg). The fact that fluoxetine can be detected in mammalian hair suggests a possible application as a monitor of drug compliance. Mean + SEM values are mg/l for blood and ng/mg for liver and hair. *P 5 0.05 when compared with hair values on day 15. Fifteen days of fluoxetine administration to rats also results in the accumulation of measurable amounts of the antidepressant in blood and liver cells. As in brain, there was significant plasma and hepatic variability in fluoxetine content between rats. Nevertheless, the drug concentrations in blood and liver are in the same range as those described by other investigators using similar spectroscopy techniques (see Table 2). Our results also confirm that plasma analytes for fluoxetine are usually lower than those measured in brain (19). Therefore, it appears that relative plasma to brain antidepressant ratios are skewed to brain, making this organ a larger depository than body fluids for fluoxetine. In contrast,
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