DILOXANIDE FUROATE A.A. Al-Majed, F. Belal and A.A. Al-Badr Department of Pharmaceutical Chemistry College of Pharmacy, King Saud University P.O. Box 2457 Riyadh- 1 145 1 Saudi Arabia ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOl.UME 26 1075-6280/00 $30.00 247 Copyright 0 1999 by Academic Press. All rishts of reproduction in any form reserved. 24X A. A. AL-MAJED ETAL. Contents 1. Description 1.1 Nomenclature 1.1.1 Chemical Names 1.1.2 Nonproprietary Names 1.1.3 Proprietary Names 1.2 Formulae 1.2.1 Empirical 1.2.2 Structural 1.3 Molecular Weight 1.4 CAS Number 1.5 Appearance 1.6 Uses and Applications 2. Method of Preparation 3. Physical Properties 3.1 X-Ray Powder Diffraction Pattern 3.2 Thermal Methods of analysis 3.2.1 Melting Behavior 3.2.2 Differential Scanning Calorimetry 3.3 Solubility Characteristics 3.4 Partition Coefficients 3.5 Spectroscopy 3.5.1 UVIVIS Spectroscopy 3.5.2 Fluorescence Spectroscopy 3.5.3 Vibrational Spectroscopy 3.5.4 Nuclear Magnetic Resonance Spectrometry I 3.5.4.1 H-NMR Spectrum 13 3.5.4.2 C-NMR Spectrum 3.5.5 Mass Spectrometry DILOXANIDE FUROATE 249 4. Methods of Analysis 4.1 Identification 4.2 Elemental Analysis 4.3 Titrimetric Analysis 4.4 Electrochemical Analysis 4.5 Spectrophotometric Methods of Analysis 4.5.1 Ultraviolet Absorption Spectrometry 4.5.2 Colorimetry Miscellaneous Non-Chromatographic Methods of Analysis 4.6 4.7 Chromatographic Methods of Analysis 4.7.1 Thin Layer Chromatography 4.7.2 Gas Chromatography 4.7.3 Supercritical Fluid Chromatography 4.7.4 High Performance Liquid Chromatography 5. Pharmacology 6. Acknowledgement 7. References 250 A. A. AL-MAJED ETAL. 1. Description 1.1 Nomenclature 1.1.1 Chemical Names [5-81 2,2-dichloro-4-hydroxy-N-methylacetanilide-2-furoate 2,2-dichloro-N-(4-hydroxyphenyl)-N-methylacet~ide-2-furoate N-dichloroacet-4-hydroxy-N-methylanilide-2-furoate 4-hydroxy-N-methy ldichloroacetanilide-2-furoate dichloro-acet-4-hydroxy-N-methylanilide-2-furoate 4-(N-methyl-2,2-dichloroacetamido)-phenyl-2-furoate 1.1.2 Nonproprietary Names Diloxanide furoate 1.1.3 Proprietary Names [5] Furamide,Histomibal,Miforon 1.2 Formulae 1.2.1 Empirical C14HllC12N04 1.2.2 Structural 0 0 1 1.3 Molecular Weight [ 5 ] 328.15 Daltons CHC12 DlLOXANlDE FUROATE 1.4 2.5 1 CAS Number 3736-8 1-0 1.5 Appearance A white or almost white, crystalline powder, which is odorless or almost odorless, and tasteless [6]. 1.6 Uses and Applications Diloxanide furoate is the furoate ester of 2,3-dichloro-4-hydroxyN-methyl acetanilide. This antiamoebic drug was developed as a result of the discovery that various a,a-dichloroacetamides possessed an amoebicidal activity [ 11. Diloxanide furoate is considered as a safe and effective drug for the treatment of asymptotic or mildly symptomatic persons who are passing cysts of Entameba histolytica [2,3]. It acts principally in the bowel lumen, and is used in the treatment of the intestinal amoebiasis. It is less effective in amebic dysentery than in asymptotic infection, but the furoate gives high intestinal concentrations and is possibly more effective than metronidazole in the treatment of cyst passers [4]. Diloxanide furoate has been used in the treatment of the asymptotic carriers of Entameba histolytica [1,3,47], and is excellent amoebicide for cyst passers [48,49]. The combination of diloxanide furoate and metronidazole in tablets is widely used for the acute and the chronic amoebiasis and giardiasis [21]. The drug is given orally in a dose of 500 mg, three times daily, for a period of ten days. Children may be given 20 mg/kg body weight, daily in divided doses, for ten days [4, 81. The drug is remarkably free of side effects, but occasional flatulence, abdominal distension, anorexia, nausea, vomiting, diarrhea, pruritis, and urticaria may occur [ 13. 2. Method of Preparation Diloxanide was first prepared by the reaction of 4-hydroxy-N-methyl aniline with sodium cyanide and chloral hydrate in the presence of a base [9]. Furoic acid was prepared by the hypochlorite oxidation of Diloxanide Iol 8 NaOCl G / C\ - O H 6 soc12 Qc-c, t; Furoyl chloride h o y l chloride 0 H D ~ l ' i J - 6 - C H C 1 2 ______) or k i c acid CH3 Qg-oe 0 r;J-C-CHClZ II CH3 Diloxanide filroate Scheme 1 Synthesis of diloxanide furoate. DILOXANIDE FUROATE 253 furfuraldehyde at low temperature [ 101. Furoyl chloride was then prepared by the reaction of furoic acid with sulfonyl chloride. The ester was in turn obtained by the condensation of diloxanide with furoyl chloride [ 1 11. The synthetic method just described was modified by Pant et al. [12], in order to eliminate the handling of furoyl chloride, and to allow the interaction of furoic acid with diloxanide. The entire procedure is illustrated in Scheme 1. 3. Physical Properties 3.1 X-Ray Powder Diffraction Pattern The x-ray powder diffraction pattern of diloxanide furoate is shown in Figure 1. This pattern was obtained using a Philips PW-1710 diffractometer, equipped with a single crystal monochromator and which used copper K a radiation. Values of the observed scattering angles, computed d-spac,ings,and relative intensities were automatically obtained on a Philips digital printer, and are given in Table 1. 3.2 Thermal Methods of analysis 3.2.1 Melting Behavior The melting point of diloxanide furoate has been reported to be 1 14-116°C [61. 3.2.2 Differential Scanning Calorimetry The differential scanning calorimetry thermogram of diloxanide furoate is shown in Figure 2. The data were obtained using a DuPont TA-9900 thermal analyzer system interfaced with the DuPont data unit. The thermogram was recorded using a heating rate of 1O"C/minute, over a temperature interval of 100-250°C. A single melting endotherm was observed having an onset temperature of 113.6"C, and a peak maximum of 114.6OC. Integration of this thermal feature indicated an enthalpy of fusion equal to 105.6 J/g. 10 15 20 25 30 35 Scattering Angle (deg. 2-8) Figure 1. X-Ray powder diffraction pattern of diloxanide furoate. 40 DILOXANIDE FUROATE 255 Table I Crystallographic Data from the X-Ray Powder Pattern of Diloxanide Furoate Scattering Angle (degrees 28) d-Spacing (A) Relative Intensity (I/Imax * 100) 6.613 10.114 11.194 13.072 13.520 13.860 14.989 16.093 17.520 17.839 18.804 19.393 20.01 1 21.525 22.189 23.03 1 23.720 24.053 24.707 25.088 25.454 25.812 27.195 13.3550 8.7382 7.8980 6.7673 6.5438 6.3844 5.9056 5.5029 5.0578 4.9682 4.7151 4.5734 4.4335 4.1249 4.0029 3.8585 3.7479 3.6968 3.6004 3.5467 3.4841 3.4487 3.2764 1.97 8.09 17.26 2.16 9.66 39.83 2.36 11.96 8.20 33.49 4.85 5.05 100.00 3.06 9.15 5.1 1 25.39 94.46 2.26 2.58 19.64 14.43 3.26 A. A. AL-MAJED ET AL. 256 Table 1 (continued) Crystallographic Data fiom the X-Ray Powder Pattern of Diloxanide Furoate Scattering Angle (degrees 20) d-Spacing (A) Relative Intensity (I/Imax * 100) 27.750 28.506 28.916 29.436 30.223 30.799 32.230 32.628 33.664 34.158 34.772 35.357 35.812 36.265 36.893 37.679 38.649 39.169 40.612 4 1.460 43.690 44.866 3.2121 3.1287 3.0852 3.0319 2.9547 2.9007 2.7751 2.7422 2.6601 2.6227 2.5779 2.5365 2.5053 2.4751 2.4344 2.3854 2.3277 2.2980 2.2196 2.1762 2.0701 2.0186 34.83 7.55 28.67 4.02 5.44 9.62 9.78 9.58 13.20 12.68 1.37 5.50 5.76 3.26 6.71 3.00 1.31 3.89 4.16 3.53 2.89 1.38 DILOXANIDE FUROATE 251 113 .58k 105.6J19 I I 105 115 I 125 1 135 Ternperature (OC) Figure 2. Differential scanning calorimetry thermogram of diloxanide furoate. 258 3.3 A. A. AL-MAJED ET AL. Solubility Characteristics Diloxanide furoate is very slightly soluble in water, soluble to the extent of 1 in 100 in alcohol, 1 in 25 of chloroform, and 1 in 130 of ether [7]. 3.4 Partition Coefficient No partition coefficient data have been reported for diloxanide furoate, but a theoretical log P value of 1.42 k 0.55 has been obtaining using the predictive program of Advanced Chemistry Development Laboratories [501. 3.5 Spectroscopy 3.5.1 UVNIS Spectroscopy The ultraviolet absorption spectrum of diloxanide furoate was recorded on a Shimadzu model 1601 PC UV/VIS spectrophotometer, and is shown in Figure 3. In aqueous solution, the spectrum exhibited a single absorption maximum located at 260 nm. For this band, the Al%:lcmvalue was 700, and the molar absorptivity equal to 22970. Clarke [7] has reported that in aqueous, the absorption maximum is observed at 262 nm, and that Al%:lcrn = 224. In ethanol, the absorption maximum is slightly shifted to 258 nm, and Al%:lcm= 705. 3.5.2 Fluorescence Spectroscopy The fluorescence spectrum of diloxanide furoate in aqueous methanol (concentration of 8 pg/mL) was recorded using a Perkin Elmer NPF-44B fluorimeter system. As shown in Figure 4, the excitation maximum was noted at 240 nm, which agrees well with the absorption maxima observed in the UVNIS studies. The emission maximum was found at 335 nm. 3.5.3 Vibrational Spectroscopy The infrared absorption spectrum of diloxanide furoate is shown in Figure 5, and was obtained as a KBr disc using a Perkin Elmer infrared spectrophotometer. The principal peaks were observed at energies of DILOXANIDE FUROATE 259 ,'-\ I # \ \ i \ i I \ I I J I I I b I I I I I I L L I I I I I I I f I I I I I I I / I / I I I / / I I I I I I I \ \ I \ I \ \ i \ \ \ \ \ I # 240 260 '--- 7 - 280 Wavelength (nm) Figure 3. Ultraviolet absorption spectrum of diloxanide firoate. A. A. AL-MAJED ETAL 260 . 240 280 320 360 400 440 Wavelength (nm) Figure 4. Excitation (EX) and emission (EM) fluorescence spectra of diloxanide furoate. 3500 --- 3000 2500 2000 1800 1600 1400 1200 Energy (cm-') Figure 5. Infrared absorption spectrum of diloxanide furoate. 1000 800 A. A. AL-MAJED ET AL 262 1678, 1 197, 1093, 1290, 1727, and 1 167 cm-'. The same absorption bands were reported by Clarke [7]. 3.5.4 Nuclear Magnetic Resonance Spectrometry 3.5.4.1 1 H-NMR Spectrum The proton NMR spectrum of diloxanide furoate was obtained using a Bruker system operating at 300,400 or 500 MHz. Standard Brucker software was used to execute recording of DEPT, COSY, and HETCOR spectra. The sample was dissolved in CH30H-d4, and all resonance bands were referenced to the tetramethylsilane internal standard. The 'H-NMR spectra are shown in Figures 6 and 7, while assignments for the observed resonance bands are given in Table 2 together with an atom numbering system. 3.5.4.2 "C-NMR Spectrum The 13C-NMRspectrum of diloxanide furoate was obtained using a Bruker system operating at 75, 100 or 125 MHz. The sample was dissolved in CH30H-d4, and all resonance bands were referenced to the tetramethylsilane internal standard. The I3C-NMR spectrum is shown in Figure 8, and assignments for the observed resonance bands are given in Table 3 along with an appropriate atom numbering system. 3.5.5 Mass Spectrometry The mass spectrum of diloxanide furoate was obtained using a Shimadzu PQ-5000 mass spectrometer, with the parent ion undergoing collision with helium carrier gas. The mass spectrum is shown in Figure 9, and features a base peak at m/z = 95, a molecular ion peak at m/e = 328, as well as numerous other fragments. Table 4 summarizes the proposed mass fragmentation pattern. Clarke reported that the principal MS peaks are found at m/z values of 95, 327, 39, 329,96, 122,244, and 67 [7]. 263 12 10 8 6 4 2 Chemical Shift (ppm) Figure 6. Full 'H nuclear magnetic resonance spectrum of diloxanide furoate. 264 II A. A. AL-MAJED ETAL %B v I I 7.6 7.4 II 7.2 I 7.0 I 6.8 I 6.6 Chemical Shift (ppm) Figure 7. Expanded 'H nuclear magnetic resonance spectrum of diloxanide furoate. 265 DILOXANIDE FUROATE Table 2 Assignments for the 'H-NMR Resonance Bands of Diloxanide Furoate Proton ID Multiplicity Chemical shift (ppm) HI doublet 7.630 - 7.634 H2 multiplet 6.535 - 6.546 H3 doublet 7.321 - 7.328 H4 and H5 two doublets 7.275 H6 and H7 two doublets 7.278 H8 singlet 3.238 H9 singlet 5.838 Atom numbering scheme for the 'H-NMR spectral assignments: EZO si - N - u zv u - c=+ rn z-5 . . . : . ” w “ “ “ “ . ‘ I ’ Chemical Shift (ppm) Figure 8. I3Cnuclear magnetic resonance spectrum of diloxanide furoate. I 0 0 v) 50 0 100 7 150 “ 0 0 0 v) 7 0 0 N 200 aI 261 DILOXANIDE FUROATE Table 3 Assignments for the I3C-NMR Resonance Bands of Diloxanide Furoate Carbon ID Chemical shift (ppm) c1 148.15 c2 139.43 c3 143.63 c4 150.68 c5 164.17 C6 128.77 c7 120.61 C8 112.86 c9 123.97 c10 112.86 Cll 120.61 c12 39.03 C13 156.68 C14 64.04 Atom numbering scheme for the I3C-NMR spectral assignments: 2mfro 5 3 1 0 0 CH3 12 14 ;di 100 3 121 I 24 3 263 I 200 . 291 310 1339 358 I 300 399 400 m/z Figure 9. 4 3 6 6 5 5 474 530 5 4 9 t Mass spectrum of diloxanide hoate. 500 DlLOXANlDE FUROATE Table 4 Fragmentation Pattern in the Mass Spectrum of Diloxanide Furoate m/Z Relative intensity Fragment 328 243 25% 124 12% 122 32% 96 55% 95 100% 67 82% Ql+ 269 A. A. AL-MAJED ETAL 270 4. Methods of Analysis 4.1 Identification Clarke has reported that diloxanide furoate gives a blue color with the Folin-Ciocalteu Reagent, and also yields a yellow color when subjected to Libermann’s test [7]. The British Pharmacopoeia has described three identification tests [6]. a. The infrared absorption spectrum of the substance is in agreement with the reference spectrum of diloxanide furoate. b. The light absorption of the substance in the range 240 to 350 nm of a 0.0014% w/v ethanolic solution exhibits a maximum only at 258 nm. The absorbance at this maximum is about 0.98. c. 20 mg of the substance is burned by the method for oxygenflask combustion, using 10 mL of 1 M sodium hydroxide as the absorbing liquid. Upon completion of the process, the liquid is acidified with nitric acid and silver nitrate solution is added. A white precipitate is produced. 4.2 Elemental Analysis [5] C: 46.18% H: 3.88% C1: 30.29% N: 5.98% 0: 13.67% 4.3 Titrimetric Analysis The British Pharmacopoeia describes a non-aqueous titration method for the determination of diloxanide furoate [6]. To perform the test, one dissolves 0.3 g of diloxanide furoate in 50 mL of anhydrous pyridine, and titrates to a potentiometric end point using 0.1 N tetrabutylammonium DILOXANIDE FUROATE 27 1 hydroxide. Each milliliter of tetrabutylammonium hydroxide titrant is equivalent to 0.03282 grams of diloxanide furoate. 4.4 Electrochemical Analysis Roy and Prakash have developed a conductimetric method for the determination of diloxanide furoate [ 131. The drug undergoes hydrolysis on heating with aqueous sodium hydroxide, and the excess sodium hydroxide is then determined conductimetrically. The method is reported to be useful for the analysis of the raw material, as well as for the analysis of tablets. 4.5 Spectrophotometric Methods of Analysis 4.5.1 Ultraviolet Absorption Spectrometry Srinath and Bagavant reported a spectrophotometric method for the analysis of binary mixtures of diloxanide furoate and tinidazole or metronidazole [14]. The mixtures were analyzed at 258 and 3 10 nm, and it was found that Beer's law was obeyed over the range of 10 to 25 pg/mL of tinidazole and diloxanide furoate. Analyte recoveries were in the range of 98.2 to 101.9%. Talwar et al. reported a simultaneous spectrophotometric determination of diloxanide furoate and metronidazole in dosage forms [ 151. The drug substances were extracted from tablets with methanol, and the extract diluted with 0.01 M sodium hydroxide. The absorbance of the solution was measured at 247 and 320 nm against 0.01 M sodium hydroxide, and the concentration of each individual drug was calculated by the Vierordt method. Drug recoveries were in the range of 99 to 1 OO%, and the method was satisfactorily applied to the analysis of commercial samples. Sethi et al. reported the assay by two methods of diloxanide furoate and tinidazole in combined dosage forms, [16]. One of these was a dualwavelength spectrophotometric method, and the other a difference spectrophotometric method. In the first method, the absorbance of sample solution was measured at 259 and 3 1 1 nm. The concentration of tinidazole was calculated from absorbance at 3 1 1 nm, and the concentration of diloxanide furoate was calculated with the use of a given equation. In the second method, the absorbance of an aqueous solution of 272 A. A. AL-MAJED ET AL. diloxanide furoate was measured at 267 nm against an alkaline solution of the drug, and also at 320 nm against an acidic solution of the drug. The analyte concentrations were then calculated from given equations. Gala1 et al. determined diloxanide furoate and metronidazole in twocomponent tablets using first (Dl) and second (Dz) derivative spectrophotometric methods [ 171. The methods were based on the direct measurement of diloxanide furoate in 0.1 N hydrochloric acid solution at 262 nm (D1 method) and at 248 nm (Dz method), without any interference from the co-existing component. The methods were applied for the determination of diloxanide furoate in the laboratory-made mixtures and in tablets, and the authors reported a relative standard deviation less than 2%. Daabees determined the drug and its degradation product by second (D2) and third (D3) derivative spectrophotometry [181. The methods were based on measuring the D2 and D3 amplitudes at 260 and 270 nm, respectively, for analytes dissolved in 0.1 N hydrochloric acid. No interference with the degradation product was noted. Das and Haider described a simultaneous spectrophotometric method for the analysis of binary dosage form mixtures of diloxanide furoate with metronidazole or with tinidazole [ 191. Powdered tablets or suspension, equivalent to 50 mg of the drug substances, were dissolved in 50 mL of dimethylformamide with shaking. After 15 minutes, the solution was diluted to 100 mL with water and filtered. A 1 mL portion of the filtrate was diluted to 50 mL with water, and the absorbance of the resulting solution measured at 320 and 262 nm for metronidazole and diloxanide furoate simultaneously. Alternatively, readings were taken at 3 I8 and 262 nm for the simultaneous determination of tinidazole and diloxanide furoate. Recoveries were reported to be quantitative. Parimoo and Umapathi used difference spectroscopy for the simultaneous quantitative determination of the drug and tinidazole in tablets [20]. The method comprised measurement of the absorbance at 282 and 240 nm of a solution of the tablet extract in pH 2 buffer solution relative to that of an equimolar solution in pH 13 buffer. Parimoo et al. have also reported the use of difference spectroscopy for the simultaneous quantitative determination of diloxanide furoate and DILOXANIDE FUROATE 213 metronidazole in a tablet preparation [21]. This study described the pHinduced difference spectrophotometric method for the quantitation of the drugs and their excipients in the presence of each other. The difference absorption of the drug solution showed maximum values of AA at 297 and 243 nm, and a minimum value of AA at 268 nm. Sanghavi and Kulkarni estimated the drug in the presence of its degraded products by differential spectroscopy [22]. Sastry et al. determined diloxanide furoate by a spectrophotometric method [23]. Two differential spectrophotometric methods were used by Chatterjee et al. for the simultaneous analysis of diloxanide furoate and metronidazole in pharmaceutical formulations [24]. The first method involved measurement of the absorbance of a methanolic solution of the two drugs at 259 and 3 1 1 nm. Since the absorbance of diloxanide furoate at 3 1 1 nm is zero, the concentration of metronidazole is directly measured, and a simple equation based on absorbance ratios is used to calculate the concentration of diloxanide furoate. The second method was a differential spectrophotometric determination based on pH-induced spectral changes, on changing from an acidic to an alkaline solution. A marked bathochromic shift was exhibited by metronidazole, while diloxanide furoate showed a slight hypsochromic shift. The wavelength of maximum absorption difference for diloxanide furoate was 267 nm, where metronidazole did not absorb. Similarly, diloxanide furoate did not interfere with metronidazole at when measured at 322 nm. Podder et al. used a simple and convenient spectrophotometric method for the determination of tinidazole and diloxanide furoate in combined pharmaceutical formulations [25]. The absorbance of a methanolic solution of a sample containing 20 to 40 pg/mL of each drug was measured between 254 to 3 10 nm. Diloxanide furoate was reported to exhibit an absorption maximum at 254 nm. Sadana and Gaonkar described a simultaneous derivative spectroscopic method for the determination of diloxanide furoate and tinidazole in pharmaceutical dosage forms [26]. Drugs were powdered and dissolved in methanol, and the solution set aside for 30 minutes with frequent shaking. After filtration, the filtrate and washings were diluted with methanol. A suspension equivalent to 150 mg of diloxanide furoate was extracted with chloroform. The filtered extract was evaporated to dryness, and the A. A. AL-MAJED ETAL. 274 residue diluted to 100 mL methanol. The extract was further diluted to 1: 10 and 1:25 with methanol, and the second derivative spectra recorded from 190 to 400 nm. 4.5.2 Colorimetry Shah and Mehta described a calorimetric method for the estimation of diloxanide furoate in pharmaceutical formulations [27]. The method is based on its interaction with hydroxylamine in alkaline solution. This method was used for the determination of the drug either alone or when combined with other agents. Diloxanide furoate was determined by Sane et al. using a simple spectrophotometric method [28]. The drug was extracted from tablets with ethanol, or was filtered from a suspension and dissolved in ethanol. The resulting solution was mixed with 6% aqueous sodium hydroxide and Folin-Ciocalteu reagent, or with a 1% solution of sodium nitroprusside in aqueous 10% sodium hydroxide. The complexes formed had absorbance maxima at 650 nm, or at 675 nm, respectively. Sastry and Aruna described the use of 3-methyl-2-benzothiazolinone hydrazone hydrochloride as a new technique for the spectrophotometric determination of diloxanide furoate and other anthelmentic and antiamoebic agents [29]. Aliquots of sample solutions containing 10-100 pg of drug substance were transferred into a series of 10 mL graduated test tubes, and the volume adjusted to 3 mL with the respective solvent blank. 3-Methyl-2-benzothiazolinonehydrazone hydrochloride and 1 mL of Cr(V1) were added, and the mixture diluted with methanol. The absorbance at 500 nm was measured against a reagent blank. Sastry et al. used iodine and isonicotinic acid hydrazide for the spectrophotometric determination of diloxanide furoate in tablets and in syrups [30]. Powdered tablets or syrup were dissolved in methanol and hydrolyzed under reflux with dilute hydrochloric acid. The mixture was cooled, and excess HCI removed under vacuum. The hydrolysate was dissolved in and diluted with water. Iodine solution and the isoniazid solution were added at two minute intervals to a potassium hydrogen phthalate/HCl buffer solution (pH 3), and diluted with water. The solution was set aside for 10 minutes, whereupon the absorbance was measured at 630 nm against a reagent blank. DILOXANIDE FUROATE 275 Sanghavi et al. reported a colorimetric method for the estimation of diloxanide furoate [3 I]. 4.6 Miscellaneous Non-Chromatographic Methods of Analysis Mohamed reported the use of proton nuclear magnetic resonance spectrometry for the assay of diloxanide furoate in bulk and in tablet formulations [32]. A solution containing 100 to 130 mg of the drug in 2 mL of trichloroethylene (also containing 12 to 14 mg/mL of tetramethylsilane internal reference) was subjected to NMR analysis. The resonances bands at 0.00 ppm (reference) and at 3.23 ppm (analyte) were each integrated three times. One gram of powdered tablets was extracted with chloroform (4 x 5 mL), the combined extracts diluted to 25 mL with chloroform, and a 15 mL portion of this solution evaporated on a waterbath. The residue was dried in vacuo over P205 and then dissolved as before, but this time in 1.5 mL of solvent, for the NMR analysis. The average drug recovery was reported to be 98%. The method of standard additions could also be used if necessary. Mohamed et al. reported the use of a flow injection procedure for the analysis of the drug among other antheimentics and antiprotozoal compounds [33]. The ethanolic sample solution (24 pL), prepared from tablets or suspensions, was injected into a carrier stream of ethanol and the absorbance measured in an 80 pL flow cell having a path length of 1 cm. The paper reports the analyzing wavelength, flow rate, and other parameters are reported. 4.7 ChromatographicMethods of Analysis 4.7.1 Thin Layer Chromatography Clarke described three thin layer chromatography (TLC) systems for the separation of diloxanide furoate [7]. Method 1 [34] Plate: Silica gel G, 250 pm, dipped in or sprayed with 0.1 M methanolic potassium hydroxide, and dried 216 4. A . AL-MAJED E T A L Mobile Phase: Developing Agent: Rf: Reference Compounds: 100: 1.5 methanol : strong ammonia solution Acidified iodoplatinate spray 66 diazepam (Rf= 7 9 , chlorprothexine (Rf= 56), codeine (Rf = 33), atropine (Rf = 18) Method 2 [34] Plate: Mobile Phase: Developing Agent: Rf: Reference Compounds: Silica gel G, 250 pm, dipped in or sprayed with 0.1 M methanolic potassium hydroxide, and dried 75: 15: I 0 cyclohexane : toluene : diethylamine Acidified iodoplatinate spray 16 dipiponone (Rf = 66), pethidine (Rf = 37), desipramine (Rf = 20), codeine (Rf = 06) Method 3 [34] Plate: Mobile Phase: 90: 10 chloroform : methanol Developing Agent: Acidified iodoplatinate spray Rr: Reference Compounds: 4.7.2 Silica gel G, 250 pm, dipped in or sprayed with 0.1 M methanolic potassium hydroxide, and dried 74 meclozine (Rf = 79), caffeine (Rf = 5 8 ) , dipipanone ( R f = 33), desipramine (Rf= 1 1 ) Gas Chromatography Clarke has described a gas chromatography system for the determination of diloxanide furoate [7]. The column is a 2 m x 4 mm internal diameter glass column containing 2.5% SE 30 on 80-100 mesh Chromosorb G (acid washed and dimethyldichlorosilane-treated). It is essential that the column DILOXANIDE FUROATE 211 be used only for performance of this method. The column temperature is 242OC, and the reference compound can be an n-alkane having an even number of carbon atoms. The retention index was reported to be 2420 WI. Sane et al. reported the determination of diloxanide furoate in pharmaceuticals by gas chromatography [36]. A sample of powdered tablets equivalent to 250 mg of drug was dissolved in chloroform and diluted to a concentration of 5 mg/mL. A mixture of 2 mL of the sample solution and 1 mL of bromhexine hydrochloride solution (the internal standard) was diluted to 5 mL with chloroform, was used for the analysis. The injection volume was 400 nL, which was analyzed at 265°C on a stainless steel column (3 m x 2 mm) containing 3% OV- 13 on Chromosorb W-I-IP (80-100 mesh). Nitrogen was used as the carrier gas at a flow rate of 50 mL/min, and analyte detection was effected using a dual flame ionization detector. Sadana and Gaonkar have simultaneously determined diloxanide furoate and tinidazole in pharmaceutical dosage form by gas liquid chromatography [371. Powdered tablets or suspension formulations were dissolved in chloroform, the solution filtered, and then diluted to 25 mL with chloroform. The solution also contained metronidazole as an internal standard. A 600 nL aliquot was analyzed on a stainless steel column (1 m x 3.2 mm) containing 3% of OV-17 on Chlorosorb W-UP (100-120 mesh). The GC system was operated at 2OO0C, using nitrogen as the carrier gas (45 mL/min). Flame ionization detection was used to observe the analytes. 4.7.3 Supercritical Fluid Chromatography The separation and estimation of diloxanide furoate and metronidazole in solid dosage forms was reported by Bhoir et al., using packed column supercritical fluid chromatography [38]. A JASCO column (10 pm particle size, 25 cm x 4 mm) was used at 40°C, with an injection volume of 20 pL. The mobile phase consisted of 26% methanol in COz (flow rate of 2 mL/min), and operated at a pressure of 17.6 MPa. When detected on the basis of its ultraviolet absorbance at 230 nm, the retention time for the drug was 1.6 minutes. The linear region of the calibration graph was reported to be 20-70 pg/mL. A. A. AL-MAJED E T AL 278 4.7.4 High Performance Liquid Chromatography Ray used high performance liquid chromatography to estimate diloxanide furoate and tinidazole in single and combined dosage forms [39]. Tablets were dissolved in the mobile phase, and 20 pL was injected on to a stainless steel column (30 cm x 3.9 mm) of p-Bondapak (218. 8:3 methanol : 0.05 M phosphoric acid (pH 3 ) was used as the mobile phase (flow rate of 2.5 mllmin), and detection was on the basis of the UV absorption at 254 nm. El-gizawy reported the analysis of diloxanide furoate in its dosage forms by a HPLC method [40]. Furazol tablets containing 200 mg of metronidazole and 250 mg of diloxanide furoate were treated with 50 mI, of methanol, sonicated for 10 minutes, and diluted to 100 mL with methanol. A portion of the resulting solution was centrifuged, and a 20 pL portion of the clear supernatant solution diluted to 10 mL with the mobile phase. This process yielded a final analyte concentration equivalent to 5 pg/mL. 20 pL aliquots of the solution were annualized by HPLC using a stainless steel column (10 cm x 4.6 mm) packed with Cyclobond I. The mobile phase consisted of 13:7 0.05 M phosphate buffer (pH 7) :methanol (flow rate of 1 mL/min), and detection was performed at 254 nm. Rao et al. reported a high performance liquid chromatographic method to determine diloxanide furoate and metronidazole in single and in combined dosage forms [41]. A 30 mg equivalent of diloxanide furoate and 25 mg of metronidazole (either as the bulk drug substances or in powdered tablets) was dissolved in methanol, amidopyrine added as the internal standard, and the mixture analyzed by HPLC at room temperature. The analytical column (30 cm x 3.9 mm) consisted of p-Bondapak CI8,with 9:9: 1: 1 methanol : water : 0.05 M KH2P04 : 0.05 M NaH2P04 as the mobile phase. The flow rate was 1 mL/min), and detection was performed at 254 nm. 5. Pharmacolow Diloxanide is used as the furoate ester, which is more effective as an antiamoebic agent than is diloxanide itself. The ester is hydrolyzed in the intestine, and only diloxanide appears in the plasma from which it is DILOXANIDE FUROATE 219 rapidly cleared [42]. The drug is well absorbed, and is present in serum largely as the glucuronide conjugate that is almost entirely excreted in the urine [ 1,431. The drug has a direct amoebicidal action, affecting the ameba before encystment [2]. 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