Claims:We claim the following from our invention,
Claim:
1. A facile spectrofluorimetric method is developed for Etodolac
a) The fluorescence spectra of the drug is being recorded as it shows native fluorescence in aqueous medium and β-cyclodextrin
b) The analysis of Etodolac in its authentic form and various available brands
c) The validation of all parameters pertaining to ICH guidelines is performed
d) Further application to weight variation testing as per USP guidelines and analysis in Human Urine is done for biological evaluation
2. As mentioned in claim 1, the excitation wavelength and emission wavelength in both aqueous and host molecule is 282 nm; 353 nm and 283 nm; 359 nm with 2.26 fold fluorescence enhancement
3. According to claim 1, the developed analogy is precise, accurate, selective and robust
4. As per claim 1, weight variation testing is being passed with accepted value 15 for individual tablets
5. As per claim 1, our invention proves that Etodolac can be quantitatively and precisely be recovered from spiked human urine samples
6. As per claim 1, the enhancement in fluorescence can be supported by calculated quantum yields with reference to Anthracene
7. As per claim 1, our invention proves that the stiochiometry between drug and host molecule is 1:1 from jobs variation plot , Description:Field of Invention
The present invention relates to the need for pharmaceutical sector to analyse micro samples of drugs that opens window for spectrofluorimetry. The whole method concentrates completely on their unique fluorescent nature besides qualitative application of spectrofluorimetry is few and major applications in drug analysis consider the assay of drugs quantitatively, metabolites and degradation products. Further quantitative estimation, facileness, consistent and specificity are best advantages of this method.
Background of the invention
Etodloac is a drug with multiple activities like antipyretic, anti-inflammatory and analgesic properties. It ellicits its response by inhibiting prostaglandin synthesis their by reducing fever, pain and swelling (Gouda et al.2006). It follows hepatic metabolism and renal excretion (Stricmann abd Balschke 2001). Over the past decade the reported methodlogies includes electrochemical (Yilmaz et al. 2001), high performance liquid chromatography (Saleh et al. 2009;Pirkle and Ficarra et al.2011; Caccamese 1992; Lee et al. 1988; Patel et al. 2001; and Ficarra et al. 1991), thin layer chromatography (Lalla et al. 1999 and Sane et al. 1998) and sequential injection analysis (Garcia et al. 2006).The described official methods are liquid chromatographic and non- aqueous titration methods for the estimation of etodolac in bulk and dosage forms (USP 27 2004; EP 2002; BP 2001 and IP2010).Various ultraviolet visible spectrophotometric methods using different derivatisation reactions (Gouda and Hassan 208; Hu et al. 2008; Ye et al. 2009;Duymus et al. 2006 and Amer et al. 2005) has been reported.
Only very few spectrofluorimetric methods for etodolac using sensitising effect of europium probe and derivatisation with 7- Fluoro- 4- Nitrobenzo- 2- Oxa-1,3 – diazole and other method coupled with spectrophotometry has been developed earlier (Eikousy et al.1999; Eihay et al. 2012 and Ulu et al. 2011).Cyclodextrins are the macromolecules that cause charesteristic luminescence enhancement by forming inclusion complexes through host guest interactions in various analyte molecules which cause considerable changes in photolytic properties of guest molecules (Szejtil 1982, Balzani and Kempet al. 1991, Kadri et al. 2004, Dhaoli et al. 2009 and Bensouilah and Abdaoui 2012).This approach has been well utilised for analytical purpose in different fluorescenceapplications of cyclodextrins ( Kinoshitha et al. 1974 -76). A significant enhancement in fluorescence response using cyclodextrins has been observed in various analytes (Santana et al. 2006, Maragos et al. 2007, Chiavaro et.al 2001,Gazpio et al. 2003 and Meras et al. 2007), drug molecules and pesticides (Sun et al. 2007, Diez et al. 2007,Walash et al. 2006, De et al. 2006 and Liu and Tang et al. 2004) coupled with other techniques. Based on the above approach for the first time to the best of our knowledge a facile macro molecule enhanced estimation of etodolac by spectrofluorimetry in spiked human urine and various tablets has been developedand further extended for weight variation test of various available brands of etodolac.
Research findings on the anti-inflammatory drug Etodolac shows that it exists as racemic mixture where S-Eto shows anti-inflammatory and R-Eto shows anti cancer property (WO01/06990). In optically pur for it has good useful properties an tis can be improved by means of incorporating cyclodextrin units with the drug through self-assembly approach. The specific action of Etodolac binding with cyclodextrins for myelosis is being known (WO01/06990). The cyclodextrin conjugated with anti-inflammatory properties along the site-specfic targeted medicaments is described in Euorpean pharmacopeia. (EP1860110B1). The extended released formulation of etodolac tablets are most widely used due to their good thereapeutic activity by Ranbaxy labs. (WO2001/019349)

The objective of present invention is to ensue a new sensitive Spectrofluorimetric method for the quantification of Etodolac in presence of _-Cyclodextrin medium. Estimate the drug in its pharmaceutical tablets and biological matrix like Human Urine and application to weight variation testing.
Summary of the invention
A facile, selective, economical and quick spectrofluorimetric method have been validated and developed for estimation of Etodolac by simple preparation steps avoiding labourious procedures with immediate fluorescence enhancement by using β – Cyclodextrin which is beneficial. Neverthless the developed strategy is sucessfuly employed for quantification of etodolac in pharmaceuticals, spiked human urine with extenstion towards weight variation test which is part of pharmacoepial requirement. Hence the proposed methodology is promising to quality control and many research laboratories with great advantage and less tedious steps.
Detailed description of the invention
An authentic Etodolac of 10 mg was used to prepare the stock solution of 100 μg/mL after sonication for 15 min in double distilled water, Followed by preparation of 5 μg/mL working solution for the further analysis with bidistilled water. Various small portions of ETO working standard solution of range 100 – 2000 μL were transferred to volumetric flask of 5 mL to get concentration range 100 – 2000 ng/mL and made up with bidistilled water. The charecteristic fluorescence intensity of ETO was recorded at 355 nm (λex = 282 nm).
Various small portions of ETO working standard solution of range 100 – 2000 μL were transferred to volumetric flask of 5 mL to get concentration range 100 – 2000 ng/mL followed by prior addition of 500 μL of β – cyclodextrin and made up with bidistilled water. The charecteristic fluorescence intensity of ETO was recorded at 359 nm (λex = 283nm). In a mortar ten tablets of different brands were first weighed, pulverised and mixed well. Accurate content of powdered Etodolac tablet of mass equal to 10 mg is taken in series of 100 mL volumetric flask followed by addition of 60 mL bidistilled water sonicated for 20 min to make sure of full removal of drug and then filtered. Working concentration range solutions (100 – 2000 ng/mL) were prepared in 5 mL volumetric flasks and methodology under section 2.4 is adapted for further analysis.
Four different brands of ETO were choosen by following representative USP guidelines 10 tablets were weighed individually, average weight is calculated and analysed by following methodology under section Methodology. 0.5 mL of diluted human urine free from drug was poured into set of centrifuge tubes and aliquots of Etodolac within the working concentration range (100 – 2000ng/mL) were added. Then Acetonitrile of volume 5 mL was being added for deprotonating and vortexed for 5 minutes. Then procedure under section 2.4 was followed by repeating the analysis for three times.
ETO Figure 1 a,b exhibits native fluorescence in aqueous media and macro molecule β-Cyclodextrin medium . There was a slightly red shift of 4 nm followed by 2.26 fold increase in fluorescence response which is due to host guest interaction of macromolecule and drug by formation of stable inclusion complex by β-cyclodextrin with Etodolac which protects the lowest excited singlet state thereby hindering the free rotational movement of molecules and preventing other non-radiative process. Moreover it can be expected that the the last ring of benzene with ethylene chain inside the β-CD

cavity with amino group at middle. The carboxylic acid group and lactone oxygen are at the first circular rim of β-CD.
Entire optimizations have been performed at room temperature (25 ̊ C ± 2 ̊ C)adapting 1000 ng/mL ETO solution. Various organised media including surfactants like Triton X – 100, CTAB, SDS and a macromolecule β – CD each 0.5 mL were careffully studied. It was evident that only the β – CD caused notable enhancement in fluorescence intensity and remaining all surfactants caused a remarkable decrease in fluorescence intensity so the β – CD has been choosen as a fluorescence enhancer for the entire analysis as shown in (Fig.2a).
Dilution with various solvents (Fig.2b) were attempted using DDW, methanol, ethanol, acetonitrile, DMSO and DMF with β – CD. A good red shift with hyperchromic effect has been observed by DDW hence the solvent of choice for analysis has been choosen to be DDW with high fluorescence intensity and lowest blank reading. Where as the protic solvents ethanol and methanol caused slight decrease in fluorescence response, on the other side aprotic solvents acetonitrile, DMSO and DMF caused sharp and distinct decrease in fluorescence signal with drug and β – CD inclusion complex. In an attempt to study concentration effect various concentrations were tested to determine optimum value for inclusion complexation and as shown in (Fig. 3a) 0.5 % w/V was choosen as the optimum concentration since at 0.4 % w/V the fluorescence intensity reached a stable level.
Two solutions of ETO and β – CD (1× 10-6 M) were prepared individually of same concentration to a total standard volume of 5 mL to study the stoichiometry of inclusion complex adapting continuous variation Job method50 followed by measuring emission at 359 nm with excitation at 282 nm of all the prepared solutions and the fluorescence intensity was plotted against ETO/ETO + β – CD. From (Fig. 3b) it is evident that the maximum fluorescence intensity was at 0.5 molar ratio from which it can be concluded that 1:1 ratio stoichiometry of ETO: β – CD is followed. The complex stoichiometry proves that the free rotational movement of drug molecule restricted giving a rigid structure to it and thereby causing enhancement in fluorescence properties which hinders other non radiative process to be taken place by energy loss to the surroundings and although the interaction here is merely hydrophobic between neutal moiety of dug molecule and macromolecule.
Satisfactory linearity has been achieved by employing a wide concentration range of 100 -2000 ng/mL by the proposed spectrofluorimetric methods. The results are illustrated followed by statiscal analysis of regression data. The precision of the developed study is evaluated by taking three concentrations, three replicates and three analysis separately over wide concentration range. Results are illustrated in Table II. The accuracy has been assesed by adapting a comparison test known as students t- values test and F- test which exhibited no significant difference between developed and comparison method.
The selctivity has been assesed as interference study from various additives in pharmaceutical tablets and studied method showed good selectivity in various brands of etodolac tablets. Robustness of the developed analysis was known by deliberate and small altering in β- Cyclodextrin volume (0.5±0.2 mL) which have no effect on FI with good stability along with experimental conditions. The weight variation test is being performed by adapting official USP guidelines for four different brands of etodolac tablets and test has been passed with its accepted value within limit of 15 with ease of greater sensitivity of developed method.
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The proposed analogy is directly tested for direct estimation of ETO in spiked samples of human urine with high recovery percentages without any prior sample derivitisation and extraction protocals with simple deprotonation step with ease of simplicity and greater sensitivity which reduces overall time consumed.
The quantum yield of ETO was calculated by using comparative method given by Horiba Jobin Vyon. A number of diluted solutions of ETO (in β-Cyclodextrin and aqueous medium) and Anthracence as reference standard were prepared in five different concentrations (100 – 1000 ng / mL) and their absorbance and integrated fluorescence intensity were recorded at 283 nm and 282 nm excitation wavelength and a graphs (Fig.4a,b,c) are plotted by taking Absorbance on x-axis and Integrated fluorescence intensity on y-axis, slope is obtained from regression equation and quantum yield is being calculated. In Ethanol Anthracence Ø=0.27, in Aqueous Medium Ø=0.27and in Triton X – 100 Medium Ø=0.67.
(NOTE: No living Organism and Microorganism is being used in this invention.)
7 Claims & 4 Figures
Brief description of Drawing
In the figures which are illustrate exemplary embodiments of the invention.
Figure 1 Drug structure of Etodolac and Fluorescence spectra of Etodolac
Figure 2 Effect of confined Media and Effect of various solvents
Figure 3 Effect of concentration and Jobs plot of Etodolac
Figure 4 Linearity plot of Anthracene; Linearity Plot of Eto in Aqueous Medium; Linearity plot of Eto in β-
Cyclodextrin medium
Detailed description of the drawing
As described above the present invention relates to development of an augmented spectrofluorimetric method to determine Etodolac in pharmaceuticals and real biological matrix.
The fluoromax-4 spectrofluorimeter is used to record the spectra of Etodolac based on its drug structure it exhibits native fluorescence shown in Figure 1.
The effect of organized media and various solvents is being studied for the developed analogy pertaining to Figure 2.
The concentration of etodolac required for forming a stiochiometric ratio of drug and host molecule like β-Cyclodextrin is shown in Figure 3.
The calculated Quantum Yield from linear plot of Anthracene, ETO in Aqueous medium and ETO in β-Cyclodextrin medium is shown in Figure 4.