ОПТИКА И СПЕКТРОСКОПИЯ, 2014, том 116, № 1, с. 57-60
СПЕКТРОСКОПИЯ ^^^^^^^^^^
КОНДЕНСИРОВАННОГО СОСТОЯНИЯ
УДК 543.42
SPECTROFLUORIMETRIC DETERMINATION OF NATEGLINIDE IN PURE AND PHARMACEUTICAL PREPARATIONS THROUGH DERIVATIZATION WITH 4-CHLORO-7-NITROBENZO-2-OXA-1,3-DIAZOLE © 2014 г. Ayga Karasakal* and Sevgi Tatar Ulu**
* Department of Chemistry, Faculty of Science and Letters, Namik Kemal University,Tekirdag, Turkey ** Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul University, 34416 Istanbul, Turkey
Received May 6, 2013
A sensitive method was developed for the determination of nateglinide in pure and pharmaceutical preparations. The proposed method is based on the derivatization reaction between nateglinide and 4-chloro-7-ni-trobenzo-2-oxa-1,3-diazole in borate buffer of pH 10. Fluorescent intensity of derivative was measured at 537 nm using an excitation wavelength of 464 nm. The described method was validated and the analytical parameters of linearity, limit of detection, limit of quantification, accuracy, precision (intra- and inter-day) and recovery were evaluated. The assay was linear over the concentration range of 50—500 ng/mL. The proposed method was applied to study of nateglinide in pure and pharmaceutical preparations.
DOI: 10.7868/S0030403414010024
INTRODUCTION
Nateglinide (NAT) is chemical name ^-(trans-4-isopropylcyclohexyl-carbonyl)-D-phenylalanine [ 1]. Nateglinide is an oral antihyperglycemic agent used for the treatment of non-insulin-dependent diabetes mellitus [2].
Some analytical methods have been described for determination of NAT in biological fluids. High performance liquid chromatography (HPLC) with UV detection have been reported for the determination of NAT in rabbit plasma and human plasma [3, 4]. HPLC method with fluorescence detection has studied of NAT in human plasma [5]. NAT and metabolites have been analysed using liquid chromatogra-phy/tandem mass spectrometry method for the determination in rat plasma [6]. Liquid chromatographic-electrospray ionization mass spectrometric method has been developed for the determination of NAT in human plasma [7].
Micellar electrokinetic chromatography method was used for the determination of NAT in animal plasma [8].
A few analytical methods have been developed for determination of NAT in pharmaceutical preparations. High performance thin layer chromatography (HPTLC) method was used for the determination of NAT in pharmaceutical dosage form [9]. NAT was analyzed using UV spectrophotometric method for the determination in bulk and pharmaceutical preparations [10, 11].
In this study, a new spectrofluorimetric method has been developed for the determination of NAT in pure and pharmaceutical preparations. The method is based on the derivatization of NAT with 4-chloro-7-
nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). NBD-Cl is a useful derivatizing agent used for the determination of amino acids, primary amines and secondary amines. In literature research, NAT has been deriva-tized for the first time by a NBD-Cl and has been determined by spectrofluorimetry.
EXPERIMENTAL
Reagents
NAT pure sample was kindly provided Deva (Istanbul, Turkey). Starlix® 120 mg film tablet was obtained from a local pharmacy. NBD-Cl was purchased from Sigma Aldrich (Steinheim). Other chemicals were provided from Merck (Darmstadt, Germany). All solvents were of analytical grade.
Apparatus
A model of RF-1501 spectrofluorimeter from Shi-madzu (Kyoto, J) was utilized to record the fluorescence spectrum of derivatized NAT-NBD compound.
Linearity range, regression equation, correlation coefficient, LOD and LOQ (n = 3)
Parameters
Linearity range, ng/mL 50-500
Regression equation F = 1.31C + 168.5
Correlation coefficient 0.9984
LOD, ng/mL 15.14
LOQ, ng/mL 45.42
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AYÇA KARASAKAL, SEVGI TATAR ULU
Cl
KM
NH
HO' "O Nateglinide
N
O
N NO2
NBD-Cl
KM
+ HCl
HO O
N
\
o
N
no2
Nateglinide-NBD derivative
Fig. 1. The reaction between nateglinide and NBD-Cl.
Solutions
Stock solution of NAT was prepared in the concentration of 1 mg/mL, in methanol. Standard solutions of NAT of 100 ^g/mL was prepared by appropriate dilution of the stock solution with methanol. A 1.5 mg/mL NBD-Cl solution was daily prepared in methanol. Borate buffer, pH 10.0 was prepared by dissolving 0.620 g boric acid and 0.750 g potassium chloride in 100 mL of water. The pH was adjusted with 0.2 M sodium hydroxide solution and the volume was made up to 200 mL with water.
General Analytical Procedure
Aliquot of NAT standard solution covering the concentration range of 50—500 ng/mL were transferred into a series of 12 mL stoppered tubes. Then, borate buffer (200 |L) and NBD-CI solutions (250 |L) were added. The solution was mixed vigorously and kept in a water bath at 70 °C for 30 min. The tube was then cooled and 100 |L of 0.1M HCl was pipetted to prevent the condensed compound from the hydrolization. The NBD derivatives were extracted three times with 2.0 mL of dichloromethane. The combined organic phases were adjusted to 10 mL with
Fluorescence intensity 1000
500
620
Wavelength, nm
Fig. 2. Emission spectrum of the reaction of (500ng/mL) NAT with NBD-Cl.
the dichloromethane. The fluorescence intensity of the NBD-derivative was measured at 537 nm after excitation at 464 nm.
Procedure for Tablets
Ten tablets of NAT were accurately weighted and the average weight of tablet was calculated. The tablets were crushed well to a fine powder. A portion of the powder equivalent to 100 mg NAT was transferred into a 100-mL calibrated flask and dissolved in 50 mL of methanol. The contents of the flask were sonicated for 30 minutes and then completed to volume methanol. The contents were mixed well and filtered; the first portion of the filtrate was rejected. An aliquot was used for the determination of each drug according to the procedure mentioned above.
RESULTS AND DISCUSSION Derivatization
NBD-Cl has a highly labile chloride atom, which is displaceable by certain nucleophile groups such as thiol, primary, and secondary amines [12—14]. NAT contains a secondary aliphatic amino group that was found to react with NBD-Cl in an alkaline buffer medium yielding a highly yellow fluorescent product (NAT—NBD derivative) that exhibited its highest fluorescence intensity at 537 nm after excitation at 464 nm (Figs. 1 and 2).
Optimization of the Reaction Conditions
The optimum reaction conditions between NAT and NBD-Cl were studied. Experimental parameters affecting the reaction were determined, such as NBD-Cl volume, pH, derivatization reaction temperature and time and diluting solvent.
Effect of Volume of NBD-Cl
The effect of the volume of NBD-Cl was studied using different volumes (25-300 |L) of 0.15% (w/v)
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SPECTROFLUORIMETRIC DETERMINATION
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reagent solution. The maximum fluorescence intensity was obtained with 250 ^L (Fig. 3).
Effect of pH
The effect of pH of the borate buffer on the deriva-tization reaction of NAT with NBD-Cl was studied over the range of 8.0—10.5. Maximum fluorescence intensities were achieved at pH 10.0 (Fig. 4).
The Effect of Temperature and Time
The effect of temperature on the derivatization reaction was also investigated from 50 to 70°C. The maximum fluorescence values were attained at 70°C within 30 min (Fig. 5).
Fluorescence intensity 600
400
200 -
0 100 200 300
^L added of 0.15% NBD-Cl
Fig. 3. Effect of the volume of NBD-Cl on the derivatization reaction.
The Effect of Diluting Solvents
The effect of diluting solvents on the fluorescence intensities of the NAT—NBD derivative was examined using different solvents methanol, ethyl acetate, chloroform, and dichloromethane. Using dichloromethane as diluting solvent gave the highest fluorescence intensities.
METHOD VALIDATION
Precision and Accuracy
NAT—NBD derivative was assayed at three concentration levels, as described under the general analytical procedure. Assays were repeated three times within the same day to determine the intra-day precision and three times on three different days to determine the inter-day precision of the method. Precision and accuracy of the method were expressed by relative standard deviation (RSD%) and relative mean error (RME%), respectively. The RME (%) and RSD (%) were found to be —0.38 % and 2.45% indicate the high accuracy and precision of the spectrofluorimetric method.
Linearity
Calibration plots were constructed by plotting the NAT concentration against fluorescence intensity. The results are shown in table.
The equations of the calibration curves are as in the followings:
F = 1.31C - 168.5 (r = 0.9984),
where Fis fluorescence intensity and C is NAT concentration (ng/mL).
Fluorescence intensity 800 -
400
9 10 pH
Fig. 4. Effect of pH on the derivatization reaction.
Fluorescence intensity 1000
500
20
40
60
Time, min
Fig. 5. Effect of temperature and time on the derivatization reaction.
0
0
Limit of Detection and Limit of Quantification
The limit of detection (LOD) and limit of quantification (LOQ) were calculated according to the current ICH guidelines [15]. LOD and LOQ were calculated
from the equation of (standard deviation of inter-cept)/(slope of regression equation) by multiplying 3.3 and 10, respectively. LOD and LOQ values were calculated as 15.14 and 45.42 ng/mL, respectively.
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AYÇA KARASAKAL, SEVGI TATAR ULU
Recovery
The recovery study was performed by adding known amounts of the compounds studied to a known concentration of the commercial pharmaceutical tablets (standard addition method). The percent recovery of the added pure drug was calculated as
% Recovery = [(Cv - Cu)/Ca] x 100,
where Cv is the total drug concentration measured after standard addition; Cu is drug concentration in the formulation; Ca is drug concentration added to formulation. The mean recovery value was 95.29%.
Pharmaceutical Application
The proposed
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