A Selective LC Method for the Determination of Reboxetine in Human Plasma with Fluorescence Detection 2007, 66, S103–S107
¨ nal&, A. Olcay Sag ˘ irli, S. Mu¨ge C¸etin, S. Toker A. O Faculty of Pharmacy, Department of Analytical Chemistry, Istanbul University, 34116 Istanbul, Turkey; E-Mail:
[email protected]
Received: 8 November 2006 / Revised: 1 February 2007 / Accepted: 8 February 2007 Online publication: 21 March 2007
Abstract A rapid, simple, accurate, sensitive and reproducible high performance liquid chromatographic method for the quantitation of reboxetine (REB) in human plasma using fluvoxamine as an internal standard (IS) has been developed and validated. The method is based on derivatization with 7-chloro-4-nitrobenzofurazan (NBD-Cl). The NBD-derivatives in plasma were extracted by liquid–liquid extraction and chromatographed on a reversed phase C18 column with isocratic elution using acetonitrile and aqueous nitric acid (pH 3) solution. Calibration curve was linear over the range 2.0–200.0 ng mL)1 with inter- and intra-assay precision (RSD%) of less than 4%. The mean recovery was about 94% for REB. The applicability of the method to the plasma was also studied.
Keywords Column liquid chromatography Reboxetine quantitation Antidepressants 7-Chloro-4-nitrobenzofurazan
Introduction Reboxetine (REB), (RS)-2-[(RS)-a-(2ethoxyphenoxy)benzyl]morpholine, is a clinical antidepressant drug [1]. The therapeutic dosage of reboxetine is usually about 20 times lower than that of tricyclic antidepressants (4–8 as opposed to 150–200 mg day)1) [2]. The single-dose pharmacokinetics of reboxetine ( £ 5 mg) is linear and the terminal elimination half-life is approximately 12–16 h in Application of Separation Techniques in Turkey.
Full Short Communication DOI: 10.1365/s10337-007-0203-0
healthy volunteers [3]. The mean absolute bioavailability of reboxetine is approximately 94% [4]. Reboxetine is more than 97% bound to plasma proteins, mainly to a1-acid glycoprotein [3, 4]. Therapeutic drug monitoring of psychotropic drugs is an established tool to optimise dosing regimen of drugs with a narrow therapeutic range, such as tricyclic antidepressants, butyrophenones or clozapine [5–7]. The therapeutic importance of these drugs requires selective, rapid and accessible methods for clinical monitoring.
Several procedures were proposed for determination of reboxetine in biological samples. Heller et al. [8] studied drug stability of a number of psychoactive drugs including reboxetine using HPLC with on-line sample pretreatment by UVvis detection. Stability of plasma samples was assessed under different conditions. Decomposition of REB at the end of a 7day period at room temperature was found to be 71%. The difference between REB plasma concentrations measured directly after preparation and at the end of 6 months storage at )20 C, was only 2% [8]. Frigerio et al. [9] reported an HPLC method with fluorimetric detection for the determination of reboxetine enantiomers in human plasma after derivatization with 9-fluorenylethyl chloroformate. The same method was then used for pharmacokinetic studies in rat and dog plasma [10, 11]. In the other enantioselective HPLC method, solid-phase extraction (SPE) was used for the determination of reboxetine (R,R)- and (S,S)enantiomers in plasma [12]. Raggi et al. [13] reported two HPLC procedures using SPE for the determination of REB in plasma. One method was liquid chromatography with ultraviolet detection; and the other method was precolumn derivatization with 9-fluorenylmethyl chloroformate by fluorimetric detection. Several HPLC–UV methods were reported for the quantification of reboxetine in plasma [3, 14, 15]. In the first one, columnswitching technique was used. In the other two reports, they are more simpler but, using of low detection wavelength
Chromatographia Supplement Vol. 66, 2007 2007 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH
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increase potential for interference and reduce specificity as well as sensitivity. Another HPLC method with dual mass spectrometric (MS) detection has been published [16]. MS detection is superior to other detection techniques and the required time for the analysis is relatively short. However, MS detection is not readily applicable for many researchers since this generally requires an expensive device. In the present study a very sensitive HPLC method for the determination of REB with fluorescence detection, was developed using a simple liquid–liquid extraction and then a derivatization reaction with the reagent, 7-chloro-4-nitrobenzofurazan (NBD-Cl). The method can easily be applied to drug monitoring.
Experimental Chemicals and Reagents Reboxetine methansulfonate and its pharmaceutical preparation (Edronax) containing 4 mg of reboxetine per tablet were kindly supplied by Pfizer (Istanbul, Turkey). NBD-Cl, boric acid, potassium chloride and sodium hydroxide were purchased from Merck (Darmstadt, Germany). HPLC grade acetonitrile, methanol, chloroform, nitric acid, hexane, isoamyl alcohol and HCl solution were obtained from Merck. Water was deionized and purified by a milli-Q water purification system from Millipore (Bedford, MA, USA). Blood plasma was obtained from healthy human volunteers and collected into tubes treated with disodium EDTA as anticoagulant. Plasma samples were stored at approximately )20 C until they were analyzed.
Preparation of Stock Solutions The stock solution of REB (1 mg mL)1) (calculated as free base) was prepared and diluted with water to give standard solutions of 2.0–200.0 ng mL)1. Standard calibration samples were prepared daily by spiking 1 mL of drug-free human plasma. The working solution of IS was prepared by dissolvation in water to obtain a concentration of 1 lg mL)1. NBD-Cl solution was freshly prepared in methanol at 3.5 mg mL)1 concentration. Buffer solution was prepared as follow: 0.620 g boric acid and 0.750 g
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potassium chloride were dissolved with 100 mL of water. The pH was adjusted to 8.5 with 0.1 N sodium hydroxide solutions and the volume was made up to 200 mL with water.
HPLC System A Shimadzu (Kyoto, Japan) LC 10 liquid chromatograph consisted of a LC 10 AT solvent delivery system, a Rheodyn injection system with a loop of 20 lL. RF-1AXL fluorimetric detector was set an excitation wavelength of 476 nm and emission wavelength of 533 nm. Separation was performed on a Phenomenex C18-column; 5 lm (250 mm · 4.6 mm i.d.) with a guard column (4.0 mm · 3.0 mm i.d.) packed with the same material. The mobile phase consisting of acetonitrile—10 mM aqueous nitric acid (pH = 3) (70:30) was delivered as an isocratic elution at a flow rate of 1 mL min)1. Before use the mobile phase was degassed by an ultrasonic bath and filtered by a Millipore vacuum filter system equipped with a 0.45 lm HV filter. The data were collected and analyzed via the automation system software.
Sample Preparation and Derivatization Blood samples were collected into the tubes containing disodium EDTA and centrifuged at 4,500·g for 10 min. Plasma calibration standards were prepared at six levels by spiking 1.0 mL of human plasma with the 10.0–1,000.0 lL of aqueous standard REB solution (200 ng mL)1). The volume was brought to 2 mL with water. The sample was basified with 0.5 mL of aqueous 0.1 mol L)1 NaOH solution after the addition of 50 lL of aqueous IS (1,000 ng mL)1) solution. Then the analytes were extracted from plasma using 6 mL of n-hexane-isoamyl alcohol (98:2) and vortex-mixing for 2 min. The samples were centrifuged for 1 min at 1,500g. Five milliliter of the organic phase was transferred into another tube for evaporation at 45 C, under nitrogen. The dried extract was reconstituted for derivatization by adding 0.2 mL of the buffer solution (pH = 8.5) and 0.2 mL of methanolic reagent solution. The capped tubes were mixed on a vortex mixer for 10 s followed by heating at 70 C for Chromatographia Supplement Vol. 66, 2007
5 min. The mixture was cooled in the ice batch and acidified using 0.2 mL of 0.1 mol L)1 HCl solution and extracted with 6 mL of chloroform:hexan (5:1), after cooling. Five milliliter of the organic phase was evaporated and the residue was dissolved in 0.5 mL of mobile phase solution. The samples were filtered through a 0.2 l membrane filter before injection into the HPLC column. Plasma samples were quantified using the ratio of the peak area of analytes to that of IS. Peak area ratios were plotted against concentrations of REB and regression equations were separately calculated.
Validation Specificity
Preparation of plasma samples were processed by liquid–liquid extraction procedure and chromatographed to determine the extent to which endogenous plasma components may contribute to the peak interference at retention time of analyte and internal standard. Linearity
The samples were assayed using the method described above. Calibration graphs were prepared by plotting the peak area ratios of REB to IS versus the drug concentrations with least-squares linear regression analysis. The sensitivity was evaluated by the lower limit of quantitation (LOQ), the lowest concentration of the plasma spiked with REB in the calibration curve. The limit of detection (LOD) was determined as the lowest concentration, which gives a signal-tonoise ratio of three for REB. The quality control (QC) samples were separately prepared in blank plasma at the concentrations of 2.0, 100.0 and 200.0 ng mL)1 for plasma.
Recovery
Absolute recoveries of REB at three QC levels (2.0, 100.0 and 200.0 ng mL)1) (n = 5) were measured by comparing the peak area of the drug obtained from the plasma with peak area obtained by the direct injection of pure aqueous drug standard. The mean recovery of the drug at three QC levels (2.0, 100.0 and 200.0 ng mL)1) was calculated by comparing the concentration obtained from Full Short Communication
the drug supplemented plasma to the actually added concentration. Precision and Accuracy
Intra-day and inter-day precision and accuracy were determined in plasma samples by determining QC samples at three concentration levels (2.0, 100.0 and 200.0 ng mL)1). For intra-day assay precision and accuracy, six replicates of samples at each concentration were assayed all at once within a day. The inter-day assay precision and accuracy was determined by samples on five different days. Six replicates at each concentration were assayed per day. Stability
The stability of REB and IS standard solutions were tested at several storage conditions (room temperature for 2 weeks and 4 C for 1 month). The stability of REB-NBD and IS-NBD derivative in the extraction solvent was determined at 4 C. The freeze–thaw stability of REB in plasma samples was evaluated over three freeze–thaw cycles. Stability control plasma samples in triplicate at the levels of 2.0, 100.0 and 200.0 were immediately frozen at )20 C, and thawed at room temperature three consecutive times. After that, the samples were processed and assayed. The stability of REB in spiked plasma stored at room temperature for 24 h and )20 C for 2 weeks was evaluated as well. Long-term stability was assessed using samples stored at )20C over a period of 8 weeks.
Results and Discussion Specificity and Separation A selective and sensitive reversed-phase HPLC method has been developed for the determination of reboxetine (REB) in plasma. Fluvoxamine (IS) [b-(Aminomethyl)-4-chlorobenzenepropanoic acid] can be determined simultaneously by HPLC (similar retention time to and good separation from the REB,) therefore, fluvoxamine was chosen as IS in this study. REB and IS contain secondary amino and primary amino groups, which are known to react with NBD-Cl in alkaline medium to yield a fluorescent derivative [17]. The product showed an absorption Full Short Communication
Fig. 1. HPLC chromatograms of a a blank plasma b a plasma samples of 1 mL spiked with 100 ng of REB and 50 ng of the IS c plasma sample from a patient who was taking 4 mg day)1 of reboxetine and 50 ng of the IS
maximum at 476 nm and a fluorescence emission peak at 533 nm. The optimum conditions were investigated and it was found that the reaction proceeds quantitatively at pH 8.5, 70 C in 5 min [18]. For the sample preparation, liquid–liquid extraction procedure (LLE) was used. Different organic solutions (chloroform, diethyl ether, ethyl acetate, hexane, hexane:ethyl acetate (98:2, v/v) and hexaneisoamyl alcohol (98:2, v/v)) were tested for this purpose and evaluated in terms of best recovery and the cleanest extracts. The best recoveries were obtained using n-hexane-isoamyl alcohol (98:2, v/v). REB was adequately separated from closely eluting endogenous amino acids in the biological samples. Following this procedure, the samples were derived from Chromatographia Supplement Vol. 66, 2007
NBD reagent. Extraction of the NBDderivates from reaction mixtures with chloroform–hexan (5:1) was provided to minimize the excess and degradation products of NBD. Also the unextracted amino acids with LLE were cleaned-up in this step. The chromatographic conditions, especially the composition of mobile phase and its pH, were optimized through several trials to achieve good resolution and symmetric peak shapes of analytes as well as short run time. For this purpose, acidic mobile phase systems were used with acetonitrile or methanol at 25 C. The best results were obtained using acetonitrile—10 mM aqueous nitric acid (pH = 3) (70:30) mobile phase system. The retention times of REB—and inter-
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Table 1. Absolute and relative recovery of REB from plasma (n = 5) Plasma sample
Concentration (ng mL)1) Added
Recovery (%)
RSD (%)
(LOD) values were 0.5 ng mL)1 for plasma with a signal-to-noise ratio three.
Found (mean ± SD)
2.0 100.0 200.0
1.90 ± 0.16 93.84 ± 2.53 192.90 ± 3.38
Absolute 95.00 93.84 96.45
2.0 100.0 200.0
1.81 ± 0.17 93.90 ± 2.66 192.04 ± 2.92
Relative 90.33 93.90 96.02
Recovery 8.61 2.70 1.75 9.15 2.83 1.52
As shown in Table 1, the mean absolute recoveries of REB were of 95.01% for plasma. The mean relative recoveries of REB were of 93.42% for plasma. The mean recovery of the internal standard was found to be 82.87% for plasma. The results in Table 1 show no clear relationship between concentration and recovery.
Table 2. Intra-day and inter-day precision and accuracy of REB in plasma (n = 6) Sample
Concentration (ng mL)1)
RSD (%)
RME (%)
Added
Found (mean ± SD)
2.0 100.0 200.0
1.79 ± 0.05 94.85 ± 1.11 191.18 ± 1.47
2.62 1.17 1.47
0.10 0.05 0.04
2.0 100.0 200.0
1.80 ± 0.07 95.51 ± 3.21 192.12 ± 3.08
3.89 3.37 1.60
0.09 0.04 0.04
The values of precision and accuracy of REB are summarized in Table 2. Intraday and inter-day relative standard deviation (RSD) values were found within 1.17 and 3.89% for plasma, respectively. The results were determined analyzing the samples spiked with REB at three different concentrations. Accuracy of the method expressed as relative mean error (RME) was below 0.10%.
Intra-day
Inter-day
Table 3. Stability of REB in plasma Treatment
Recovery (mean ± SD) (%) 2.00a
Three freeze–thaw cycles Stored at RT for 24 h Stored at )20 C for 2 weeks Stored at )20 C for 8 weeks
95.59 85.74 92.56 86.00
100.0a ± ± ± ±
0.004 0.003 0.03 0.01
97.98 86.05 93.97 87.00
± ± ± ±
Stability
200.0a 0.04 0.06 0.03 0.03
98.01 86.90 94.01 86.23
± ± ± ±
0.21 0.22 0.29 0.23
RT room temperature a Plasma concentration (ng mL)1)
nal standard—NBD derivatives were 12.49 and 13.87 min, respectively and the total analysis run time was 15 min. Representative chromatograms of (a) drug-free plasma, (b) the plasma spiked with REB (100.0 ng mL)1) and internal standard (50.0 ng mL)1), (c) the plasma obtained at 2.5 h from a patient who was taking 4 mg day-1 of REB, are given in Fig. 1. There is no interference in the chromatogram of drug-free plasma. Numerous studies provide evidence that major depression is associated with certain disorders [19–21]. So, reboxetine is co-administired with the other drugs. Commonly prescribed antidepressant drugs (nortriptyline, desipramine, paroxetine, maprotiline, fluoxetine, sertraline, imipramine, risperidon, quetiapine, ziprasidon, citalopram, venlafaxine,
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Precision and Accuracy
lamotrigine, carbamazepin) were analyzed for possible interference. No interference peak was observed under the chromatographic conditions.
Calibration and Linearity Calibration curves were linear over the range 2.0–200.0 ng mL)1 for plasma. The regression equations were as follows: A = 0.0109 C + 0.0539 (r = 0.9981), where A is the peak area ratios (AREB/ AIS) and C is the concentration of REB (ng mL)1). The limit of quantitation (LOQ) values for each sample was accepted as the lowest concentration on the calibration curves which were 2.0 ng mL)1. Under the experimental conditions, the lower limit of detection Chromatographia Supplement Vol. 66, 2007
The derivative of REB-NBD was stable in this solvent for at least 48 h at 4 C in the dark. The stability of stock solutions of REB in water was checked and proved to be stable for at least 1 month at 4 C. The stock solution of IS in water was stable for 2 weeks at 4 C. The stabilities of drug and IS in biological fluid are affected by the chemical properties of drug and IS, the storage conditions, the matrix effects. The stability of REB under various conditions in described in Table 3. Under all conditions tested, REB was stable with detected concentrations of at least 85.74% for plasma of the initial concentration.
Application to Patient Plasma Sample The proposed HPLC method with fluorescence detection was applied to the analysis of plasma samples from patients (aged 59 years, weighing 63 kg), undergoing therapy with reboxetine tablets. The chromatogram of a plasma sample from a patient who was taking 4 mg day-1 of reFull Short Communication
boxetine is shown in figure. There was no interference from the matrix or other coadministered drugs. The reboxetine concentration found in this sample, obtained by interpolating on the blank plasma calibration curve, was 132 ng mL-1.
Conclusion
Acknowledgments The authors would like to thank the Research Fund of Istanbul University for support of this work (Project numbers: 416/13092005).
References
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In this study a new, very sensitive, and reliable HPLC method using fluorimetric detection has been developed for the assay of reboxetine in plasma samples. The method is based on a derivatization with 7-chloro-4-nitrobenzofurazan (NBD-Cl) reagent that is widely used to produce fluorescence derivatives of the compounds with primary and secondary amine groups [22, 23]. This reagent is cheaper than other fluorimetric reagents. The developed method is relatively simple and rapid to perform, requiring liquid– liquid extraction and one derivatization step prior to chromatography. The method showed high selectivity, precision and accuracy proving its usefulness for therapeutic monitoring of REB.
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