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 Table of Contents  
ORIGINAL ARTICLE
Year : 2012  |  Volume : 4  |  Issue : 3  |  Page : 236-245  

A novel chiral GC/MS method for the analysis of fluoxetine and norfluoxetine enantiomers in biological fluids


Department of Clinical Pharmacology and Therapeutics, The University of Malta, Msida, MSD 2040, Malta

Date of Submission23-Sep-2011
Date of Decision13-Dec-3011
Date of Acceptance20-Dec-2011
Date of Web Publication26-Jul-2012

Correspondence Address:
Janet Mifsud
Department of Clinical Pharmacology and Therapeutics, The University of Malta, Msida, MSD 2040
Malta
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-7406.99065

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   Abstract 

Aims: A novel robust chiral gas chromatographic/mass spectrometric (GC/MS) method for the separation and measurement of fluoxetine and norfluoxetine enantiomers in urine and plasma was developed. Materials and Methods: The drug was extracted from the samples by a liquid-liquid technique, using chloroform, and the enantiomers were separated and measured on a chiral gas chromatographic column (HYDRODEX β-6TBDM®, 0.25 μm × 0.25 mm × 50 m). GC/MS instrumentation was used for the acquisition of data in the electron impact selective-ion monitoring mode. Results: The ions chosen were of a mass-to-charge ratio (m/z) exactly equal to 44 units, in order to measure fluoxetine enantiomers, 134 units in order to measure norfluoxetine enantiomers, and 58 units in order to measure diphenhydramine, the internal standard. The method was found to be linear and reproducible in the 50-500 ng/mL concentration range for both urine samples and plasma samples and for both fluoxetine and norfluoxetine, with correlation coefficients ranging between 0.994 and 0.997. Conclusions: This methodology has an enormous potential for application in pharmacokinetic studies of the enantiomers of fluoxetine

Keywords: Chiral, cyclodextrin, fluoxetine, GCMS, urine


How to cite this article:
Mifsud J, Sghendo LJ. A novel chiral GC/MS method for the analysis of fluoxetine and norfluoxetine enantiomers in biological fluids. J Pharm Bioall Sci 2012;4:236-45

How to cite this URL:
Mifsud J, Sghendo LJ. A novel chiral GC/MS method for the analysis of fluoxetine and norfluoxetine enantiomers in biological fluids. J Pharm Bioall Sci [serial online] 2012 [cited 2017 Mar 29];4:236-45. Available from: http://www.jpbsonline.org/text.asp?2012/4/3/236/99065

Fluoxetine is a selective serotonin reuptake inhibitor that has been shown to exert its antidepressant effect by interacting with a membrane protein. [1] It is a chiral molecule that is commercially available as a racemic mixture, that is, a 50:50 mixture of its enantiomers, (R)-fluoxetine, and (S)-fluoxetine. (S)-fluoxetine and (R)-fluoxetine are equipotent in blocking serotonin reuptake. [2] However, the enantiomers of the demethylated metabolite, norfluoxetine, have been shown to have marked differences in the pharmacological activity, with (S)-norfluoxetine being about 20 times as potent as (R)-norfluoxetine as a serotonin reuptake inhibitor. [2]

Several methods have been described for the determination of racemic fluoxetine and norfluoxetine in human urine, plasma, serum, whole blood, hair, or environmental water. The most widely used methods involved high-performance liquid chromatography with ultraviolet, [3],[4] fluorescence, [5],[6],[7] and diode array detection. [8],[9] Fluoxetine and norfluoxetine levels have also been measured in human urine samples, human whole blood samples, pharmaceutical formulations and cellular structures using gas chromatography coupled with mass spectrometry, [10],[11],[12] flame ionization, [13] electron capture, [14] or nitrogen-phosphorus detection. [15]

However, only limited chiral work has been done on the stereoselective disposition of fluoxetine and norfluoxetine. The stereoselective disposition of fluoxetine and its main metabolite norfluoxetine has been studied in a pregnant sheep. [16] The results showed that both fluoxetine and norfluoxetine rapidly cross the placenta. Also, there was significant stereoselectivity of fluoxetine during the crossing, with the ratio of (S)-fluoxetine to (R)-fluoxetine area under the plasma concentration versus time curve ranging from 1.65 to 1.73 in the fetus, after maternal dosing. [16] However, in this case, only modest validation of the analytical technique was carried out.

The measurement of the enantiomers of fluoxetine and norfluoxetine in human plasma and serum using two-dimensional gas-liquid chromatography with nitrogen-phosphorus selective detection has also been described. [17] The enantiomers were extracted from plasma and serum using liquid-liquid extraction and injected into a GC/MS system for detection and quantification. The system incorporated two columns in tandem, one achiral and the other consisting of derivatized, beta-cyclodextrin. Nisoxetine and fluvoxamine were used as internal standards and these chemicals could have influenced negatively the results obtained since their retention times were higher than those of both fluoxetine and norfluoxetine enantiomers. Also, in this research work, the enantiomers of fluoxetine were not separated completely.

One other enantioselective investigation has been employed in the comparison of the disposition of fluoxetine and norfluoxetine enantiomers in the mother, fetus, and infant. [18] The plasma concentrations of (S)-fluoxetine and (R)-fluoxetine and of (S)-norfluoxetine and (R)-norfluoxetine were measured by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. The results showed that there was a high correlation between maternal and fetal (cord blood) fluoxetine and norfluoxetine enantiomers with the mean fetal to maternal ratios equaling 0.91 and 1.04, for fluoxetine and norfluoxetine, respectively. However, once more, validation of the analytical technique was not detailed and elaborated.

Thus, in this research work, a novel technique for the chiral gas chromatographic-mass spectrometral analysis of the antidepressant agent fluoxetine and its primary metabolite norfluoxetine was developed, based on the utilization of a chiral stationary phase consisting of cyclodextrins. Cyclodextrins (CDs) are a family of cyclic oligosaccharides, composed of 5 or more a-D-glucopyranoside units linked together in such a way so as to form a particular arrangement. [19] They have found extensive use in separation science because they have been shown to discriminate between positional isomers, functional groups, homologues, and enantiomers. This feature makes them one of the most useful agents for a wide variety of separations, including the resolution of primary and secondary amine compounds. [17],[20]

The research work carried out in our laboratory was novel in many ways. The water removal procedure was applied for the first time in order to dry our samples. Diphenhydramine was successfully employed for the first time as an internal standard in the analysis of the enantiomers of fluoxetine and its main metabolite. The 50 m chiral column utilized was employed for the first time, alone, in such an analytical technique. The temperature program utilized was just enough for the nearly baseline resolution of the enantiomers, without lengthening the chromatographic process further. Furthermore, mass spectrometry was utilized for the first time, in combination with the 50 m chiral column employed, in order to measure the enantiomers of fluoxetine and norfluoxetine.


   Materials and Methods Top


Description of chemicals

Fluoxetine, (dl)-N-methyl-3-phenyl-3-[4-(trifluoromethyl) phenoxy]-propan-1-amine, C 17 H 18 F 3 NO, is a creamish white crystalline powder with a molecular weight of 302. The chemical structure of fluoxetine is shown in [Figure 1]. The melting point of fluoxetine powder is 158°C, the pK a of fluoxetine base is 9.5 and fluoxetine crystals are soluble in methanol, chloroform, and diethylether, less soluble in water and practically insoluble in hexane and ethyl acetate. Norfluoxetine, (dl)-3-phenyl-3-[4-(trifluoromethyl) phenoxy]-propan-1-amine, C 16 H 16 F 3 NO, is a yellowish white hygroscopic powder with a molecular weight of 288 and similar physical characteristics to fluoxetine.
Figure 1: Chemical structure of fluoxetine

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The extraction of both fluoxetine and norfluoxetine from biological specimens is usually achieved with either liquid-liquid or solid-phase extraction. Diphenhydramine, 2-(diphenylmethoxy)-N,N-dimethylethanamine, C 17 H 21 NO, the internal standard, is a white or almost white, crystalline powder with a molecular weight of 255. The chemical structure of diphenhydramine is shown in [Figure 2]. The melting point of diphenhydramine powder is 168°C, its pK a is 8.3 and the crystals are soluble in methanol, chloroform, diethylether, and water.
Figure 2: Chemical structure of diphenhydramine

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Chemicals and solutions

Chloroform, anhydrous ammonium sulfate powder, sodium hydroxide pellets and anhydrous sodium sulfate powder were all of Analar® grade (BDH, Poole, England). Rac-fluoxetine hydrochloride, (R)-fluoxetine hydrochloride and rac-norfluoxetine hydrochloride, were bought from Sigma® Company (Poole, Dorset, UK). The internal standard, diphenhydramine hydrochloride and dimethoxypropane were also bought from Sigma® Company (Poole, Dorset, UK). Quantitative chromatographic analytical work cannot be performed without the use of a suitable internal standard.

There is no traditional internal standard for the gas chromatographic/mass spectrometral analysis of fluoxetine and norfluoxetine. One investigator has opted for imipramine, [21] although the structure of this compound is considerably different from that of fluoxetine and norfluoxetine. Clomipramine, 2,4-dichlorophenol, flumazenil, fluvoxamine, nortriptyline and oxazepam have also been used as internal standards for the quantitative determination of fluoxetine and of its enantiomers. [4],[14],[22],[23],[24],[25] In our research work, we opted to use diphenhydramine since its structure is relatively small and similar to that of both fluoxetine and norfluoxetine and because its retention time is smaller than that of the enantiomers, and therefore, it elutes earlier.

Stock solutions and calibration standards for both rac-fluoxetine and rac-norfluoxetine and for the internal standard diphenhydramine were prepared at adequate concentrations in order to be measured as accurately as possible in biological fluids. In urine, calibration standards were prepared at 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 ng/mL. In plasma, calibration standards were prepared at 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 ng/mL. The stock solutions and calibration standards were kept refrigerated all the time.

Equipment

The GC/MS system used in these studies consisted of a quadrupole instrument with a direct capillary column interface and an electron-ionization type ion source (GCMS-QP5050® , Shimadzu, Kyoto, Japan). The instrument was equipped with a turbomolecular pump (Turbotronik NT 151/361, Shimadzu, Kyoto, Japan), backed-up by a rotary mechanical pump (Edwards, Sussex, England). The peaks were recorded and measured with CLASS-5000 software (Shimadzu, Kyoto, Japan) on a Funai computer. The instrument operated on both a scan mode and on a selective-ion monitoring mode. The gas chromatograph (GC-17A® , Shimadzu, Kyoto, Japan) was equipped with a chiral, fused-silica, capillary column - HYDRODEX b-6TBDM®, 0.25 mm × 0.25 mm × 50 m (Macherey-Nagel GmbH and Co., Düren, Germany).

Chromatographic conditions

Extracted urine or plasma samples (1 mL) were introduced in a split mode into the injection port, maintained at 250°C. Urine samples were analyzed at a split ratio of 1:20, while plasma samples were analyzed at a split ratio of 1:10. The column head pressure was maintained at 20.31 psi using ultrapure helium as a carrier gas. The transfer line temperature was held at 250°C and the oven was temperature programmed. The column temperature was maintained at 160°C for exactly 1 min, ramped at 3°C per minute to 190°C and held at this temperature for 1 min and then ramped again at 1°C per min to 195°C. A final hold time of 30 min was set.

The mass spectrometer was operated in the electron ionization/scanning mode for the preliminary qualitative work, collecting data between 40/50 and 450 mass units (mu), at 2 scans per second. For quantitative analysis, the mass spectrometer was operated in the electron ionization/selective-ion monitoring mode, collecting ions of a mass to charge ratio (m/z) exactly equal to 44 and specific for the enantiomers of fluoxetine, collecting ions of a mass to charge ratio exactly equal to 134 and specific for the enantiomers of norfluoxetine and collecting ions of a mass to charge ratio exactly equal to 58 and specific for diphenhydramine.

Urine extraction

Urine samples were extracted using a modification of previously published procedures. [17],[22] Urine samples, 500 mL, were mixed by vortexing for 1 min with 500 mL of diphenhydramine hydrochloride, the internal standard, at a concentration of 100 ng/mL and 200 μL of 2 M sodium hydroxide solution in 17 ×58 mm, foil-face lined, bakelite screw-capped, glass vials (BDH, Poole, England). Next, 5 mL of chloroform was added and the contents were vigorously shaken on a rotary shaker for 30 min. Excess anhydrous sodium sulfate powder was then added and the contents were centrifuged for 10 min at 2800 × g.

The organic layer from each sample was later transferred to a clean vial. The remaining aqueous layer from each sample was extracted two more times with chloroform. The total chloroform layers were dried with anhydrous sodium sulfate powder overnight by shaking on a rotary shaker. Next day, the sodium sulfate powder was sedimented by centrifugation for 10 min at 2800 × g and the organic layer was transferred to a clean vial. The samples were later evaporated under a stream of air at room temperature, for approximately 15 min, to around 250 mL. Dimethoxypropane was added to the samples at the final stages of evaporation, to ensure thorough removal of water. Extracted samples (1 μL) were injected into the gas chromatographic/mass spectrometric system for analysis.

Plasma extraction

The extraction procedure used was a modification of the ones employed by other investigators. [17],[22] The thawed plasma samples, 200 μL, were mixed by vortexing for 1 minute with 200 μL of diphenhydramine hydrochloride, the internal standard, at a concentration of 100 and 50 μL of 2 M sodium hydroxide solution in 16 × 100 mm, rubber-stopper, evacuated, glass tubes (Vacutainer®, Becton Dickinson, USA). Chloroform, 5 mL, was added and the samples were vigorously shaken on a rotary shaker for 30 min. Excess anhydrous sodium sulfate powder was then added and the contents were centrifuged for 10 min at 2800 × g. The organic layer was later transferred to a clean vial.

The remaining aqueous layer from each sample was then extracted two more times with chloroform. The total chloroform layers were dried with anhydrous sodium sulfate powder overnight by shaking on a rotary shaker. Next day, the sodium sulfate powder was sedimented by centrifugation for 10 min at 2800 × g and the organic layer was transferred to a clean vial. The samples were subsequently evaporated down to approximately 50 mL under a stream of air. Dimethoxypropane was added at the end of the evaporation step. Extracted samples (1 mL) were injected into the gas chromatographic/mass spectrometric system for analysis.

Measurement of concentrations and variations

Concentrations of the enantiomers in the standard samples were calculated using peak area ratios. Peak area data were generated from the computer software controlling the instrument. The ratios obtained were used to construct calibration graphs. Linear regression and correlation coefficient were estimated for all curves using Sigma Plot® software. Intraday and interday coefficients of variation were estimated from injections of extracted standard samples of rac-fluoxetine and rac-norfluoxetine at 75 and 425 ng/mL in order that the reproducibility of the results of both the parent drug and metabolite were analyzed at the same concentrations.

Intraday variation in analysis was determined from six consecutive injections of extracted urine samples and nine consecutive injections of extracted plasma samples. Interday variation in peak area measurement was determined by injecting an extracted urine sample on six separate days and an extracted plasma sample on nine separate days. Recovery was estimated by adding exact quantities, that is, 125 and 375 ng/mL, of rac-fluoxetine and rac-norfluoxetine to blank urine (n = 6) or blank plasma (n = 9) and extracting and injecting consecutively. Statistical analysis was carried out on the data using Microsoft Excel 2008® software.

The limit of detection (LOD) of the analytical method was taken to be the lowest concentration of enantiomer that could be detected but necessarily not quantitated, under the above experimental conditions. The limit of quantitation (LOQ) of the analytical technique was defined as the concentration at which quantitative results could be reported with a high degree of confidence. Statistically, the limit of detection is defined as three times the standard deviation of the blank, while the limit of quantitation is defined as 10 times the standard deviation of the blank. Therefore, the limit of quantitation was taken to be equal to 3.3 times the limit of detection.


   Results and Discussion Top


Fragmentation data

The identical mass spectra of the enantiomers of fluoxetine and norfluoxetine, shown in [Figure 3]a and b and the mass spectrum of diphenhydramine, shown in [Figure 4], were consistent with the information available at the Mass Spectrometry Data Centre and with the results obtained by other investigators [16],[18] for fluoxetine and norfluoxetine. The major peaks for fluoxetine were detected at m/z 44, 59, 91, and 104. Other smaller peaks were detected at m/z 77, 115, 132, 148, and 164. The small peak at m/z 309 represented the molecular ion for fluoxetine.
Figure 3: Mass spectrum of (a) fluoxetine and (b) norfluoxetine. The mass spectra of fluoxetine enantiomers were identical and the major peaks were detected at m/z 44, m/z59 and m/z 104. The mass spectra of norfluoxetine enantiomers were also identical and the major peaks were detected at m/z 104 and m/z 134. No molecular ion peak was obtained for both chiral agents

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Figure 4: Mass spectrum of diphenhydramine. The major peaks were detected at m/z 45, m/z 58 and m/z 73. Again, no molecular ion peak was obtained in this case

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The principal peaks for norfluoxetine were detected at m/z 51, 77, 83, 104, and 134. Other smaller peaks were detected at m/z 143, 162, and 191. The very small peak at m/z 295 represented the molecular ion for norfluoxetine. It must be pointed out that the peak at m/z 83 originated from the solvent and not from the fragmentation of norfluoxetine. The main peaks for diphenhydramine were detected at m/z 45, 58, and 73. Other smaller peaks were detected at m/z 165 and 167. No peak at m/z 255, representing the molecular ion for diphenhydramine, appeared in this case. The mass spectral data for diphenhydramine attained in our studies was consistent with the results obtained by other researchers. [26],[27]

In the case of fluoxetine, the peak at m/z 44 occurred due to the loss of the secondary amine group, that is, CH 2 NHCH 3 group, [28] while the peak at m/z 148 resulted due to bond breakage between the asymmetric carbon atom and the oxygen atom. [29] In the case of norfluoxetine, the peak at m/z 134 resulted due to bond breakage between the asymmetric carbon atom and the oxygen atom, [28] while the peak at m/z 30, seen in some schematic representations of the fragmentation of norfluoxetine but not in our case, resulted due to the loss of the CH 2 NH 2 group. [28]

In our analysis, data were collected between 50 and 450 mass units for norfluoxetine and therefore the peak at m/z 30 was not obtained. This was done in order to reduce interference by small ion fragments. The fragmentation pattern of diphenhydramine was also identical and consistent with the information available at the Mass Spectrometry Data Centre. The peak at m/z 45 occurred due to the loss of the HN (CH 3 ) 2 group. The peak at m/z 58 resulted due to the loss of the CH 2 N (CH 3 ) 2 group. The peak at m/z 73 occurred due to the loss of the HCH 2 CH 2 N (CH 3 ) 2 group. The peaks at m/z 166 and m/z 167 resulted due to the loss of one ring from the structure and the rearrangement of the resulting molecular fragment.

Calibration analysis

The resolution of the enantiomers of both fluoxetine and norfluoxetine by the chiral gas chromatographic column was highly acceptable, as shown in [Figure 5]. (S)-fluoxetine, the first enantiomer of fluoxetine eluting according to Ulrich (2003) [17] and preliminary studies, had a retention time of 35.90 min, while (R)-fluoxetine had a retention time of 36.35 min. (S)-norfluoxetine had a retention time of 35.85 min, while (R)-norfluoxetine had a retention time of 36.50 min. Diphenhydramine, the internal standard, had a retention time ranging between 30.60 and 31.00 min.
Figure 5: (a) Chromatogram for urine samples spiked with 350 ng/mL rac-fluoxetine at m/z 44; diphenhydramine (30.60 min), (S)-fluoxetine (35.90 min) and (R)-fluoxetine (36.35 min). (b) Chromatogram for plasma samples spiked with 400 ng/mL rac-norfluoxetine at m/z 134; diphenhydramine (30.95 min), (S)-norfluoxetine (36.00 min) and (R)-norfluoxetine (36.55 min)

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These retention times were higher than those observed by Conley and his colleagues (2008) [30] and Unceta and his colleagues (2007). [29] They were also higher than the ones observed by Ulrich. [2] In his research, Ulrich (2003) [17] observed retention times of 14.90 min for (S)-norfluoxetine, 15.90 min for (R)-norfluoxetine, 15.50 min for (S)-fluoxetine, and 15.80 min for (R)-fluoxetine. The column retained its ability to separate completely the enantiomers of both fluoxetine and norfluoxetine throughout all the course of the studies.

The detector response was somewhat higher at m/z 44 than at m/z 134 and the peaks of the enantiomers were of similar dimensions compared to the internal standard peak, at m/z 44. At m/z 134, the baseline was somewhat higher sometimes and peak areas of the enantiomers were also acceptable. Peak shape was symmetrical, acceptable and adequate for peak area measurement and peaks obtained for all calibration standards, both in urine and plasma, at both m/z 44 and m/z 134, had similar shapes and retention times to those shown in [Figure 5].

The calibration graphs constructed at both m/z 44 and m/z 134, for both urine and plasma samples, were linear and with very low intercepts on both axis. Linear regressions and correlation coefficients were excellent for both enantiomers. At m/z 44, r 2 for urine samples was 0.997 [Figure 6], while for plasma samples it was 0.995 [Figure 6]b. At m/z 134, r 2 for urine samples was 0.997 [Figure 6]c, while for plasma samples it was 0.994 [Figure 6]d.
Figure 6: Typical calibration graphs for (a) urine fluoxetine samples, (b) plasma fluoxetine samples, (c) urine norfluoxetine samples and (d) plasma norfluoxetine samples and for (S)- ( and #61550;) and (R)- ( and #61543;) enantiomers. (Statistical analysis: Data points= 10; Standard Deviation: Slope= 0.00048 - 0.00053, Intercept= 0.075 - 0.082; Standard error of the mean: Slope= 0.00061 - 0.00068, Intercept= 0.0944 - 0.1056)

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The calibration graphs obtained were described by the following equations:

  1. y = 0.0080x - 0.2382 (S-); y = 0.0082x - 0.2390 (R-) [urine, m/z 44, [Figure 6]a]
  2. y = 0.0080x - 0.2519 (S-); y = 0.0080x - 0.2487 (R-) [plasma, m/z 44, [Figure 6]b]
  3. y = 0.0082x - 0.2551 (S-); y = 0.0082x - 0.2501 (R-) [urine, m/z 134, [Figure 6]c]
  4. y = 0.0081x - 0.2664 (S-); y = 0.0081x - 0.2610 (R-) [plasma, m/z 134, [Figure 6]d]
where y was peak area ratio, while x was enantiomer concentration.

The calibration graphs confirmed that the levels of the enantiomers of both fluoxetine and norfluoxetine, in calibration standards, were within the limits of detection. These results compared very well with those obtained by Ulrich (2003), [17] where the correlation coefficient for plasma samples was found to be equal to 0.995 for (S)-fluoxetine and 0.994 for (R)-fluoxetine, while a value of 0.995 was obtained as the correlation coefficient for both (S)-norfluoxetine and (R)-norfluoxetine.

In his research, Ulrich (2003) [17] calibrated both the enantiomers of fluoxetine and the enantiomers of norfluoxetine and obtained acceptable values for both precision and accuracy of the method. The very low intercepts on both axis showed that the graphs nearly passed through the origin. The results obtained above also compared well with those obtained by other researchers, such as Gatti and his colleagues (2003), [31] who used an enantioselective column consisting of 3,5-dimethylphenylcarbamate as a chiral selector coated on 10 μm silica gel to resolve the enantiomers of fluoxetine and the enantiomers of norfluoxetine, respectively.

Intraday and interday variation

Variation in intraday and interday analysis was determined by calculating the coefficients of variation. As shown in [Table 1], in urine samples at m/z 44, the intraday coefficient of variation range between 2.17% and 3.25%, while the interday coefficient of variation ranged between 2.86% and 4.03%. At m/z 134, intraday variation was never lower than 3.51% and never higher than 4.42%, while interday variation was never lower than 5.03% and never higher than 6.26%.

Considering intraday urine samples at m/z 44, the standard deviation ranged between 0.48 and 0.86, while in the case of intraday urine samples at m/z 134, the standard deviation had a lowest value of 0.39 and a highest value of 1.10. Focusing on interday urine samples at m/z 44, the standard deviation was in the range of 0.66 and 1.17, while a similar trend occurred in interday urine samples at m/z 134, where the standard deviation was never lower than 0.55 and never higher than 1.60.
Table 1: Intraday and interday variation for urine samples at m/z 44 and m/z 134

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Coefficients of variation were again calculated for plasma samples. As illustrated in [Table 2], in plasma samples at m/z 44, intraday variation ranged between 2.55% and 3.39%, while interday variation ranged between 4.16% and 5.26%. At m/z 134, the intraday coefficient of variation was never lower than 4.66% and never higher than 6.42%, while the interday coefficient of variation was never lower than 6.21% and never higher than 11.70%.
Table 2: Intraday and interday variation for plasma samples at m/z 44 and m/z 134

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Furthermore, taking into consideration intraday plasma samples at m/z 44, the standard deviation ranged between 0.43 and 2.22, while in the case of intraday plasma samples at m/z 134, the standard deviation had a lowest value of 0.65 and a highest value of 0.86. Focusing on interday plasma samples at m/z 44, the standard deviation was in the range of 0.95 and 1.26, while a similar trend occurred in interday plasma samples at m/z 134, where the standard deviation was never lower than 0.72 and never higher than 2.41.

Overall, the intraday and interday coefficients of variation were low and less than 12%. Intraday variation for urine samples was higher at m/z 134 than at m/z 44 and higher for the (S)-enantiomers than for the (R)-enantiomers. Also, variation at 75 ng/mL was higher for the (S)-enantiomer and lower for the (R)-enantiomer than variation at 425 ng/mL. Interday variation for urine samples was usually higher than intraday variation and basically more pronounced at m/z 134 than at m/z 44. Moreover, in interday analysis at m/z 44 and m/z 134, variation at the lower concentration, that is, 75 ng/mL was not always higher than variation at the higher concentration, that is, 425 ng/mL.

Intraday variation of plasma samples at m/z 44 and at m/z 134 was usually higher than variation in urine. A similar trend occurred in the case of plasma samples, with a frequently higher variation at m/z 134 than at m/z 44, a higher or lower variation at 75 ng/mL than at 425 ng/mL and a higher variation for the (S)-enantiomers than for the (R)-enantiomers. Interday coefficients of variation were the highest in these studies, although still relatively low (<12%). In fact, interday variation in plasma at 425 ng/mL was only slightly higher than variation at the same concentration in urine (~1.50%). Only the coefficients at 75 ng/mL varied to a considerable extent from urine to plasma and from intraday to interday analysis. Ulrich (2003) [17] reported in a similar way such an increase in the coefficient of variation from urine extracted samples to plasma extracted samples and from intraday to interday measurements.

Recovery

Recovery rates in the extraction of urine and plasma samples at both m/z 44 and m/z 134 were similarly determined by calculating the coefficients of variation. As shown in [Table 3], in urine samples at m/z 44, mean recovery was high and in the range of 95.46% to 97.23%, while the coefficient of variation ranged between 2.77% and 4.55%. At m/z 134, mean recovery was in the range of 95.00% to 96.49%, while the coefficient of variation was never lower than 3.51% and never higher than 5.00%. In plasma samples at m/z 44, mean recovery was again high and ranging from 94.88% to 96.77%, while the coefficient of variation ranged between 3.23% and 5.12%. At m/z 134, mean recovery was in the limits of 91.68% to 94.42%, while the coefficient of variation was never lower than 5.57% and never higher than 8.32%.
Table 3: Recovery data for urine and plasma samples at m/z 44 and m/z 134

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Considering recovery data for urine samples at m/z 44, the standard deviation had a lowest value of 0.89 and a highest value of 1.19. In the case of recovery data for urine samples at m/z 134, the standard deviation had a lowest value of 1.13 and a highest value of 1.78. Focusing on recovery rates for plasma samples at m/z 44, the standard deviation was never lower than 1.54 and never higher than 2.30. Similarly, in the case of recovery rates for plasma samples at m/z 134, the standard deviation was never lower than 0.74 and never higher than 1.01. All in all, the above results compared well with those obtained in 2007 by Unceta and his colleagues. [29]

Unceta and his colleagues (2007) [29] obtained recovery rates ranging between 92.30% and 94.00% for the enantiomers of fluoxetine and between 91.20% and 92.90% for the enantiomers of norfluoxetine. Our results suggested that recovery rates of the enantiomers of both fluoxetine and norfluoxetine from biological fluids were very high and in the range of 91% and 98%. The coefficients of variation, although low, were usually higher in plasma than in urine and usually higher at m/z 134 and at 125 ng/mL for both plasma and urine. One exception occurred at m/z 44 and at 375 ng/mL, where variation was higher in urine than in plasma for both (S)- and (R)- enantiomers. The highest variation in recovery data was obtained in plasma, at m/z 134 and at 125 ng/mL, where variation exceeded 8.00%. All in all, the extraction procedure employed seemed reliable and reproducible.

Limit of detection and limit of quantitation

The limit of detection of the analytical technique was similar for both urine and plasma samples, at both m/z 44 and m/z 134 and was essentially equal to 3.20 ng/mL of the enantiomers of fluoxetine and 4.20 ng/mL of the enantiomers of norfluoxetine. Consequently, as described in the methodology, the limit of quantitation of the developed method was also similar for both urine and plasma samples, at both m/z 44 and m/z 134 and was equal to 10.50 ng/mL of the enantiomers of fluoxetine and 12.50 ng/mL of the enantiomers of norfluoxetine.

Ulrich (2003) [17] obtained a value of 1.5 ng/mL of (R)-fluoxetine and (S)-fluoxetine and a value of 6 ng/mL of (R)-norfluoxetine and (S)-norfluoxetine for the limit of detection of their analytical technique. Djordjevic and his colleagues (2005) [22] measured a value of 2.5 ng/mL of (R)-fluoxetine and (S)-fluoxetine and a value of 10 ng/mL of (R)-norfluoxetine and (S)-norfluoxetine for the limit of quantitation of their analytical method.

The internal standard peak did not coelute with the peak of each enantiomer or with peaks generated by any other compound coextracted from the samples. The retention times obtained in these studies, that is, 35.85 minutes for (S)-norfluoxetine, 36.50 min for (R)-norfluoxetine, 35.90 min for (S)-fluoxetine, and 36.35 min for (R)-fluoxetine, were acceptable.

In fact, such retention times were longer than the times that had been previously reported with another successful chiral chromatographic separation of rac-fluoxetine and rac-norfluoxetine, that is, 14.90 min for (S)-norfluoxetine, 15.90 min for (R)-norfluoxetine, 15.50 min for (S)-fluoxetine, and 15.80 min for (R)-fluoxetine. [17] However, in the latter case, a 15 m achiral column connected in series with a 25 m chiral cyclodextrin column were employed in the analysis. On the other hand, calibration analysis, intraday/interday variation and recovery data obtained in our studies were similar to those observed by the same researcher and by one other research group. [17],[29]

The main striking feature of our results was that although in our research work only one column was employed, a HYDRODEX b-6TBDM®, 0.25 mm × 0.25 mm × 50 m (Macherey-Nagel GmbH and Co., Düren, Germany), the method was still powerful enough to obtain a highly satisfactory resolution of the enantiomers of fluoxetine and norfluoxetine. Furthermore, the peak shapes and sizes obtained were adequate for the accurate measurement of the enantiomers. This meant that the temperature program utilised in our research work was just enough to compensate for the absence of an achiral column in our system, without compromising severely the run time.

The chiral stationary phase used in our research and consisting of heptakis-(2,3-di-O-methyl-6-O-t-butyldimethylsilyl)-b-cyclodextrin diluted in optimised polysiloxane proved to be efficient in separating the enantiomers of both fluoxetine and norfluoxetine to an acceptable extent. It has been well documented that derivatized b-cyclodextrin shows the broadest enantioselectivity towards different guest molecular classes. [32] Regioselective derivatization of the hydroxyl groups in cyclodextrins allow more attractive interactions, such as hydrogen bonding, to take place where previously the guest molecule would have projected well beyond the edge of the underivatized cyclodextrin. [33]

The stability of the cyclodextrin molecule is enhanced by complete or partial substitution of the hydroxyl functions on the parent molecule. On the substitution of these hydroxyl groups, the conformation of the cyclodextrin system is altered and this has a great influence in improving the selectivity towards the guest molecule. [34] It is thought that inclusion complexation between the cyclodextrin molecule and the guest molecule may occur more rapidly with the high temperatures used in gas chromatography than with the lower temperatures used normally in liquid chromatography. Thus, better chiral resolution may be achieved with cyclodextrin chiral stationary phases used in gas chromatography. [35]


   Conclusion Top


A novel modified, reproducible GC/MS method for the analysis of fluoxetine and norfluoxetine enantiomers in urine and plasma was developed. The chiral stationary phase was successful in separating the enantiomers, although the retention times obtained were somewhat long. No contaminating compounds interfered with the elution of the peaks originating by the internal standard or the enantiomers. Fragmentation patterns for fluoxetine, norfluoxetine and diphenhydramine were within the parameters described by international organizations. Calibration analysis was acceptable and peak area ratios were found to have a highly linear relationship with the concentrations of the standards at all instances. Also, intraday/interday variation and recovery statistics proved that the analysis was a reproducible one. The method developed offers an enormous potential for application in determination of fluoxoetine in biological samples for pharmacokinetic studies.


   Acknowledgements Top


Funding was obtained through research grants awarded by the University of Malta.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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