Etodolac methods

This report presents various methods developed primarily at our laboratory for chromatographic resolution of racemates of several pharmaceuticals (e.g., -blockers, NSAIDS, anta-acids, DL-amino acids, Bupropion, Baclofen, Etodolac, Carnitine, Mexiletine). Recently, we developed methods for establishing molecular dissymmetry and determining absolute configuration of diastereomers (and thus the enantiomers) of (/< .S )-Baclofcn, (/d.SJ-Bctaxolol with complimentary application of TLC, HPLC, H NMR, LCMS this ensured the success of diastereomeric synthesis and the reliability of enantioseparation. 

Rodriguez de Pablos, R., Garcia-Ruiz, C., Crego, A. L., and Marina, M. L. (2005). Separation of etodolac enantiomers by capillary electrophoresis. Validation and application of the chiral method to the analysis of commercial formulations. Electrophoresis 26(6), 1106—1113. 

A method for preparative scale separation of the enantiomers of etodolac involved crystallization of racemic etodolac with optically active 1-phenethylamine [13]. Racemic etodolac was allowed to crystallize with either S(-)- or R(+)-phenethylamine in anhydrous 2-propanol for 12 hours at 4°C. Pure diastereomeric salts were decomposed with sulfuric acid (10% v/v), and extracted with ethyl acetate. The enantiomeric purity of both enantiomers was at least 98%. 

A colorimetric method for the analysis of etodolac has been reported which is based on the formation of colored complexes with p-dimethyl-aminobenzaldehyde in the presence of sulfuric acid and ferric chloride [19]. Absorbance measurements were made at 591.5 nm, and the method was found to be linear over the concentration range of 10 to 80 pg/mL. This method was used to determine etodolac in bulk powder and other dosage forms. 

A simple, sensitive, and reproducible fluorimetric method for the determination of etodolac in bulk powder or dosage forms has been reported [19]. The method involves measurement of the native fluorescence at a wavelength of 345 nm, when ethanolic solutions of the drug were excited at 235 nm. The calibration was found to be linear over the concentration range of 96 to 640 ng/mL. 

A fluorimetric method developed for prodolic acid can also be used to quantitate etodolac in serum [20], This method involved using a 1 1 mixture of isoamyl alcohol and n-heptane to extract the drug from serum. An aliquot of the organic phase was then mixed with a 1 1 mixture of dimethyl sulfoxide and isoamyl alcohol, and the fluorescence of the clear solution was determined by excitation at 280 nm and scanning the emission spectra from 240-370 nm. The limit of detection of this method was about 2 pg/mL. 

A TLC method has been developed to determine etodolac and its metabolites (6-OH-etodolac and 7-OH-etodolac) in biological fluids and extracts (before and after enzyme hydrolysis) [15]. The method used silica gel plates and hexane-ethyl acetate-acetic acid (60 40 2, v/v) and hexane-ethyl acetate (70 30, v/v) solvent systems to separate the free carboxylic acid and methyl esters of etodolac and the two metabolites. The relative retention (Rf) values obtained under these conditions were 0.29,0.20 and 0.24 for etodolac, 6-OH-etodolac, and 7-OH-etodolac respectively. An Rf value of 0.45 was obtained for methyl ester of etodolac. 

Another TLC method has been reported, which uses 0.25 or 2 mm silica gel plates as the stationary phase, and ethyl acetate/methanol (95 5, v/v) or toluene/ethyl acetate (7 3, v/v) as the developing agent [21]. The method was used to separate etodolac and its metabolites in urine and bile. 

A sensitive reverse-phase HPLC method has been developed for the analysis of etodolac in tablet formulation [22]. The chromatographic separation was achieved using a reverse-phase Cu column, having dimensions of 3.3 cm x 0.46 cm i.d. (3 pm particles) and which was maintained at 30°C. The mobile phase consisted of pH 6.0 phosphate buffer / methanol (60 40 v/v), and was eluted at 1 mL/min. Analyte detection was effected on the basis of UV detection at 230 nm. Diazepam was used as an internal standard. The sample preparation entailed grinding the etodolac tablets, followed by extraction with methanol (using sonication). A retention time of 1.46 min was obtained for etodolac under these conditions, and the method was found to be linear, precise, and accurate over the concentration range of 0.01 to 0.1 mg/mL. 

A simple, accurate, and reproducible HPLC method has been developed to determine etodolac in presence of impurities (1-methyl and 8-methyl-etodolac) and in pharmaceutical formulations [14]. A Viospher ODS-2 (15 cm x 4.6 mm i.d., 5 pm particle size) HPLC column was used as stationary phase, and acetonitrile/0.05 M phosphate buffer (pH 4.75) (60 40 v/v) eluted at 0.8 mL/min was used as mobile phase. The system was thermostatted to 25 °C, and acetaminophen was used as an internal standard. Detection was achieved by measurement of the UV absorbance at 229 nm. The method was found to be linear over the concentration range of 2-20 pg/mL. The relative retention times for etodolac and acetaminophen were 2.2 and 2.9 min respectively. The retention times for the two impurities, 1-methyl-etodolac and 8-methyl-etodolac, were 1.4 and 3.8 min respectively. 

A stability indicating HPLC method has been developed to measure etodolac in presence of three main degradants, 7-ethyl-2-( 1 -methylene-propyl)- 1 -//-indole-3 -ethanol, the decarboxylated product of etodolac, and 7-ethyltryptophol [23]. A reverse phase ODS column (15 cm x 0.41 cm i.d., 5 pm particles) was used to achieve separation. The mobile phase... 

A simple, isocratic chromatographic method for the separation, identification, and measurement of etodolac enantiomers without derivitization using chiral stationary phase columns has been reported [25]. A chiral stationary phase column packed with Chiracel OD (cellulose tris-3,5-dimethylphenylcarbamate coated on 10 pm silica gel) was used as the stationary phase. The mobile phase (85 15 v/v, n-hexane/2-propanol (containing 0.1% trifluoroacetic acid)) was pumped at 0.7 mL/min and the UV detection was set at 230 nm. The (-)-(/ )-etodolac enantiomer eluted first, indicating its stronger interaction between the stationary phase relative to the (+)-(S)-etodolac enantiomer. 

The use of capillary electro-chromatography for the separation of etodolac and its metabolites has recently been reported [26, 27], Separation of etodolac was achieved using 3 different methods capillary HPLC, capillary electro-chromatography (CEC), and pressure assisted CEC. Fused-silica capillaries of 100 pm i.d. were packed with 5 pm octadecyl silica (Cig) under 400 bar pressure. The length of the packed capillaries was 24.5 cm. 

An isocratic HPLC method for screening plasma samples for sixteen different non-steroidal anti-inflammatory drugs (including etodolac) has been developed [29]. The extraction efficiency from plasma was 98%. Plasma samples (100-500 pL) were spiked with internal standard (benzoyl-4-phenyl)-2-butyric acid and 1 M HC1 and were extracted with diethyl ether. The organic phase was separated, evaporated, the dry residue reconstituted in mobile phase (acetonitrile-0.3% acetic acid-tetrahydrofuran, in a 36 63.1 0,9 v/v ratio), and injected on a reverse-phase ODS 300 x 3.9 mm i.d. column heated to 40°C. A flow rate of 1 mL/min was used, and UV detection at 254 nm was used for quantitation. The retention time of etodolac was 30.0 minutes. The assay was found to be linear over the range of 0.2 to 100 pg/mL, with a limit of detection of 0.1 pg/mL. The coefficients of variation for precision and reproducibility were 2.9% and 6.0%, respectively. Less than 1% variability for intra-day, and less than 5% for inter-day, in retention times was obtained. The effect of various factors, such as, different organic solvents for extraction, pH of mobile phase, proportion of acetonitrile and THF in mobile phase, column temperature, and different detection wavelengths on the extraction and separation of analytes was studied. 

A reverse-phase microbore HPLC method with photodiode-array detection and UV spectral library was developed for toxicological screening of various drugs in plasma including etodolac and its methyl ester [30]. Sample preparation involved addition of prazepam (internal standard) to 500 pL of plasma followed by addition of 30 pL of 1M sodium hydroxide and 5 mL of dichloromethane. After shaking the sample for 1 minute and centrifuging, the upper aqueous layer was discarded. The organic phase was evaporated and reconstituted with 50 pL methanol and 20 pL water ... 

An HPLC method to determine etodolac during in vitro studies was developed [31]. Plasma was precipitated with acetonitrile, evaporated to dryness, and reconstitution in 25% acetonitrile. The HPLC consisted of a guard column, a 4 x 150 mm (5 pm particles) Cig column, and UV detection at 280 nm. The mobile phase was methanol / 0.01 M trifluoroacetic acid (25 75, v/v) at 1 mL/min. Etodolac was located at a retention time of 15.2 minutes. 

An HPLC method to determine racemic etodolac and its major metabolites in urine using a reverse-phase column has been developed [13]. Determination of etodolac in urine involved acidifying the diluted urine before extraction with cyclohexane-ethyl acetate (95 5, v/v). The organic layer was separated, evaporated, and reconstituted in solution of ibuprofen in acetonitrile (internal standard). The samples were injected onto a LiChrosper 100 RP-18 25 cm x 4 mm i.d., (5 pm particle size) column. 

The mobile phase was 0.05 M pH 4 phosphate buffer / acetonitrile (55 45, v/v) at a flow rate of 1.3 mL/min. Detection was carried out at 220 nm with a UV detector. The retention times were 12.5 and 16.9 minutes for etodolac and ibuprofen, respectively. The method was linear over a concentration range of 0.125 to 10.0 pg/mL. The recovery of etodolac was 93.9 % ( 5.3 %). Detailed methods for the analysis of metabolites of etodolac were also provided. 

An HPLC method for the analysis of etodolac and its metabolites in equine serum and urine was developed [32]. Serum (1 mL) or urine (0.5 mL) samples were extracted with iso-octane/isopropanol (95 5, v/v) after addition of ibuprofen as internal standard, diluting with 1 or 2 mL of distilled water, and adjusting the pH to 1 with 1 M HC1. The organic layer was evaporated under a stream of nitrogen, the residue dissolved in 100 pL of mobile phase, and a 20 pL aliquot injected on to the HPLC system. The HPLC system consisted of a pre-column, a 250 x 4 mm (7 pm particles) LiChrosorb RP-18 column at 25°C, isocratic elution with 1% acetic acid/acetonitrile (50 50, v/v) at a flow rate of 1.3 mL/min, and UV detector at 227 nm. The retention time of etodolac was 8.5 minutes. The method was linear over the range of 0.1-20 pg/mL in serum, and in 0.5-800 pg/mL range in urine. The limits of quantitation were 40 ng/mL in... 

A GS-MS method for the analysis of etodolac in human plasma has been developed [16]. Plasma samples were spiked with meclofenamic acid (the internal standard), acidified with 5N HC1, and extracted twice with chloroform / dichloromethane / hexane (50 25 25, v/v). The organic phase was evaporated, the residue methylated with ethereal diazomethane, dried again, and reconstituted in hexane. Analytical separation was performed on a 15 m x 0.24 mm i.d., 0.25 pm film thickness, fused silica capillary column. The oven temperature was variable (150 to 260°C) and the injector was at 260°C. The carrier gas was helium at 1 mL/min. Mass spectra were obtained using positive electron impact ionization (70 eV) at m/z 228 for etodolac. The method was linear in the 1-10 ng/mL (low) and 10-100 ng/mL (high) concentration range. The detection limit was 0.5 ng/mL in plasma, and recovery of etodolac from plasma sample exceeded 92%. 

The capillary chromatography method previously described [26,27], has also been used for the analysis in the etodolac and its metabolites in urine. 

A stereoselective GC method for determination of etodolac enantiomers in human plasma and urine was first reported as a preliminary method [35], and then as a validated method [36]. Sample preparation involved addition of (S)-(+)-naproxen (internal standard) and sodium hydroxide to diluted plasma or urine. The samples were washed with diethyl ether, acidified with hydrochloric acid, and extracted with toluene. ( )-(+)-naproxen was used as a derivatizing agent to form diastereomeric derivatives of etodolac. The gas chromatograph system used in this work was equipped with fused-silica capillary column (12 m x 0.2 mm i.d.) coated with high-performance cross-linked methylsilicone film (thickness 0.33 pm) and a nitrogen-phosphorous detector. The operating conditions were injector 250°C detector 300°C column 100-260°C (32 °C/min). 

No significant stereoselectivity in etodolac enantiomeric metabolism was noted in a 24 hour urine recovery study [40]. Analytical methods for the... 

Cosyns, L. Spain, M. Kraml, M. Sensitive high-performance liquid chromatographic method for the determination of etodolac in serum. J.Pharm.ScL, 1983, 72, 275-277... 

Etodolac (ETD) is a non-steroidal anti-inflamatory antirheumatic drug. A survey of the literature reveals that there is no method available for the determination of ETD in pure form and pharmaceutical formulations by oxidation-reduction reactions. 

We describe three simple, sensitive and reproducible spectrophotometric assays (A-C) for the determination of etodolac in pure form and in pharmaceutical formulations. Methods A and B are based on the oxidation of... 

Our methods were successfully applied to the determination of etodolac in bulk and pharmaceutical formulations without any interference from common excipients. The relative standard deviations were < 0.76 %, with recoveries of99.87 % — 100.21 %. 

Etodolac (ETD), l,8-diethyl-l,3)4,9-tetrahydropyrano- [3,4-b]indole-1-acetic acid [1], is a non-steroidal anti-inflamatory antirheumatic drug (Scheme 1). A survey of the literature reveals that there are very few reported methods for the determination of ETD in biological fluids, pharmaceutical formulations and in presence of its enantiomer. Of those studies reported, the techniques used include chromatography, HPLC [2-5], GC [6-8], in addition to spectrofluorimetric [9] and spectrophotometric methods [9-11]. However, an extensive survey of the literature revealed that there is no method available for the simultaneous determination of ETD in pure form and pharmaceutical formulations by oxidation-reduction reactions. 

Etodolac (ETD) pure grade was supplied by Pharco, Egypt. The purity was found to be 100.35 0.64 % according to the Pharco method [26] in which the absorbance of 0.002% w/v etodolac solution in 0.1 N sodium hydroxide was measured at 276 nm. 

Etodolac that belongs to a class of nonsteroidal antiinflammatory drugs, used for the treatment of mild-to-moderate pain, fever, and inflammation, can be resolved by preferential crystallization after a conglomerate formation of its derivatives. Using this method, etodolac enantiomers were recovered with an overall yield of more than 20% and the purities were greater than 99.9%. Methods for the separation of enantiomers dming crystallization are discussed in chapter 56 in more detail. 

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