ОПТИКА И СПЕКТРОСКОПИЯ, 2012, том 112, № 3, с. 476-482
= СПЕКТРОСКОПИЯ КОНДЕНСИРОВАННОГО СОСТОЯНИЯ
УДК 535.243
VALIDATED SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION, SPECTROSCOPIC CHARACTERIZATION AND THERMAL STRUCTURAL ANALYSIS OF DULOXETINE WITH l,2-NAPHTHOQUINONE-4-SULPHONATE
© 2012 г. Sevgi Tatar Ulu*, Fikriye Tuncel Elmali**
*Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul University, 34416, Istanbul, Turkey **Department of Chemistry, Faculty of Arts and Sciences, Yildiz Technical University, 34210, Esenler, Turkey
E-mail: sevgitatar@yahoo.com Received May 13, 2011
Abstract—A novel, selective, sensitive and simple spectrophotometric method was developed and validated for the determination of the antidepressant duloxetine hydrochloride in pharmaceutical preparation. The method was based on the reaction of duloxetine hydrochloride with 1,2-naphthoquinone-4-sulphonate (NQS) in alkaline media to yield orange colored product. The formation of this complex was also confirmed by UV—visible, FTIR, 1H NMR, Mass spectra techniques and thermal analysis. This method was validated for various parameters according to ICH guidelines. Beer's law is obeyed in a range of 5.0—60 p.g/mL at the maximum absorption wavelength of 480 nm. The detection limit is 0.99 p.g/mL and the recovery rate is in a range of 98.10—99.57%. The proposed methods was validated and applied to the determination of duloxetine hydrochloride in pharmaceutical preparation. The results were statistically analyzed and compared to those of a reference UV spectrophotometric method.
INTRODUCTION
Duloxetine hydrochloride (DH), (+)-(^)-^-Me-thyl-3-(naphthalene-1-yloxy)-3-(thiophen-2-yl)pro-pan-1-amine hydrocloride [1], is a potent antidepressant which also acts as a central analgesic [2]. DH is a selective serotonin and norepinephrine reuptake inhibitor (SNRI) that claims greater affinity for the serotonin and norepinephrine transporters compared with venlafaxine [3—4].
Several analytical methods have been reported for determination of DH in biological fluids and in pharmaceutical preparations .These methods include liquid chromatography with single-quadrupole mass spectrometric (LC-MS) method [5], liquid chromato-graphic-tandem mass spectrometry (LC-MS-MS) [6, 7], liquid chromatography with atmospheric pressure ionization—tandem mass spectrometry [8] gas chro-matography-nitrogen-phosphorus detection (GC-NPD), gas chromatography—mass spectrometry (GC-MS) [9], HPLC [10, 11], capillary electrophoresis [12], spectrofluorimetry [13, 14] and spectrophotometry [15, 16].
NQS has been used as a chromogenic reagent for the spectrophotometric determination of primary and secondary amines [17—24]. According to the literature research, DH has been derivatized with NQS and determined using a UV-visible spectrophotometric method for the first time. In this study, a simple, rapid, accurate and sensitive UV-visible spectrophotometric method was developed to assay DH in pharmaceutical
preparations. The solid complex has been synthesized and structure of the complex has been confirmed by spectral techniques such as UV—vis, FTIR, XH NMR, mass and also by thermal analysis.
MATERIALS AND METHODS
Materials
DH was kindly supplied from Eli Lilly (Istanbul, Turkey). Cymbalta capsules (60 mg) were purchased from a local pharmacy. The NQS was purchased from (Merck, Darmstadt, Germany). All solvents used were of analytical reagent grade (Merck, Darmstadt, Germany). Water was prepared by using aquaMAX™ ultra, Young instrument (Korea) ultra water purification system.
Apparatus
UV-160A (Shimadzu, Kyoto, Japan) ultraviolet-visible spectrophotometer with matched 1 cm glass cells was used for all spectrophotometric measurements.
The FTIR spectra were recorded with ATR technique on a Perkin Elmer Spectrum One Bv 5.0 spectrophotometer. 1H NMR spectra were recorded on a Varian UNITY INOVA 500 MHz spectrometer. TMS was used as internal standard in 1H NMR. Mass spectra (LC-MS/MS) were determined on a FinniganTM LCQTM Advantage MAX spectrometer. TGA-DSC
(Thermogravimetric analysis-Differential Scanning Calorimetry) curves were obtained with a TA SDT Q 600 thermal analyzer apparatus using flowing nitrogen 100 mL/min, temperature range 25—1200°C, at heating rate of 20°C.
Preparation of Solutions
Stock solution of DH was prepared as 1.0 mg/mL in methanol. Appropriate dilutions of DH were made in methanol to produce working standard solution of 100 ^g/mL. A borate buffer (0.1 M) was prepared by dissolving 0.620 g of boric acid and 0.750 g of potassium chloride in 100 mL water. The pH was adjusted to 9.0 with 0.1 M sodium hydroxide solution and the volume was made up to 200 mL with water. NQS was prepared as a 0.5% (w/v) solution of distilled water.
Preparation of Pharmaceutical Preparation Solution
The contents of twenty capsules were weighed and mixed. A quantity of the powder equivalent to 100 mg of DH was transferred into a 100 mL volumetric flask, dissolved in methanol, and sonicated for 30 min., the volume was then completed with methanol, shaken well for 5 min. and filtered. Finally, a 10 mL aliquot of this solution was diluted to 100 mL with methanol in a volumetric flask. This solution was analyzed as in the general analytical procedure.
General Procedure
Aliquot of standard DH solution (0.25 mL—3.0 mL; 100 ^g/mL) were transferred into a series of 12 mL volumetric flasks containing 0.5 mL of borate buffer pH 9.0 and 0.5 mL of 0.5% NQS reagent solution was added in each tube and the contents were heated at 50°C for 40 min and cooled. Then, the above solutions were adjusted to 5 mL with the methanol. The absor-bances were measured at 480 nm against a reagent blank prepared in a similar way.
RESULTS AND DISCUSSION
Derivatization Conditions
NQS is a general spectrophotometric reagent for both primary and secondary amine groups. The reaction of NQS with DH that own a free secondary amine group results in the formation of coloured products (Fig. l). The product was orange-colored exhibiting a maximum absorption peak (^max) at 480 nm (Fig. 2).
The spectrophotometric properties of the colored product as well as the different experimental parameters affecting the color development and its stability were carefully studied and optimized. Such factors were changed individually while the others were kept constant. The factors include pH, type, ofbuffer, temperature and time, effect of reagent and diluting solvent.
The pH was varied over the pH range of 8—10 using borate buffer where the maximum absorbance was ob-
Absorbance
Wavelength, nm
Fig. 2. a. Duloxetine only (20 ^g/mL); b, NQS (20 p.g/mL); c. Absorption spectrum ofthe derivative of duloxetine with NQS (60 p,g/mL).
Absorbance 0.8
10.0 pH
Fig. 3. Effect of pH on absorbance of duloxetine with NQS.
Absorbance
0.8 -
0.7 -
0.6 -
0.5
0.4 -
0.3 - a-
0.2 -
0.1 i
0 10
50°C
70°C
60°C i=
Room temperature
20
30
40
50 60 Time, min
Fig. 4. Effect of time and temperature on the reaction of duloxetine with NQS.
tained at pH 9.0 as shown in Fig. 3. The effect of using different buffer solutions such as carbonate or phosphate buffers as alternatives to borate buffer at this optimum pH value was also investigated. It was found that borate buffer is better in obtaining the maximum absorbance.
The effect of temperature on the color intensity was studied by heating the reaction mixture over the range from room temperature to 70°C for different periods of time. It was that heating at relatively lower temperature (50°C) for 40 min gave more reproducible results in Fig. 4.
The influence of the concentration of NQS was studied using different volumes of 0.5% w/v of the reagent solution. It was found that the reaction of NQS with DH started upon using 0.1 mL of the reagent. 0.5 mL of 0.5% (w/v) of NQS solution was chosen as the optimal volume of the reagent.
Different solvents were tried to dilute the reaction mixture throughout the study. It was observed that methanol gave the highest absorbance. Using of chloroform, dichloromethane, chloroform:n-butanol (4:1) and dichloromethane:n-butanol (4:1) produce relatively low absorbance. The increase of absorbance due to the use of methanol may be attributed to lowering the absorbance of the blank reagent.
The stability of DH-NQ derivative was examined in terms of absorbance at the concentration of 60 ^g/mL but no change in absorbance of more than 1.5% was observed within 48 hat + 4°C.
The stoichiometric ratio of the formed product was investigated by Job's continuous variation method [25]. For the study, NQS and DH solutions, prepared as 3 x 10-4 M, were mixed in varying volume ratios in which the total volume of the mixtures was kept at 3 mL. The solutions were analyzed as in the general analytical procedure. Application of Job's method of continuous variation indicated a molar ratio of DH to NQS of 1:1 (Fig. 5).
Fig. 5. Mole ratio of NQS to DH.
FTIR Spectra
The FTIR spectral bands of the DH, NQS and DH-NQ complex are listed in Table 1. The FTIR spectrum of the DH-NQ complex is compared with the DH and NQS. The -NH stretching vibration band was observed at 3328 cm-1 in the DH. This band disappeared in the complex spectrum. Althought the aliphatic -CH2 groups appeared at 2941 and 2914 cm-1 in the DH, this bands was shifted at 2935 cm-1 in the DH-NQ complex. The band at 1673 and 1661 cm-1 was assigned to the stretching vibration of the -C=O groups of the NQS. This bands was shifted in the DH-NQ complex toward lower wave numbers (1642 cm-1). All these changes could be related to the expected electronic structure modifications in the formed complex.
Fig. 6. Mass spectra of DH-NQ complex.
Relative 100
90
80
70
60
50
40
30
20
10
abundance
476.82
100 200 300 400 500 600 700 800 900 1000
m/z
0
1H NMR Spectra
The *H NMR spectral data of the DH, NQS and DH-NQ complex are given in Table 2. The *H NMR spectrum of DH-NQ complex was compared
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