Spectrophotometric Methods for the Simultaneous Determination of Binary Mixture of Metronidazole and Diloxanide Furoate Without Prior Separation

Journal of Advanced Research (2010) 1, 323–329

Cairo University Journal of Advanced Research

ORIGINAL ARTICLE Spectrophotometric methods for the simultaneous determination of binary mixture of metronidazole and diloxanide furoate without prior separation

Mohamed R. El-Ghobashy a,*, Nisreen F. Abo-Talib b a Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt b National Organization for Drug Control and Research (NODCAR), Cairo, Egypt

Received 26 October 2009; revised 1 March 2010; accepted 18 March 2010 Available online 30 June 2010

KEYWORDS Abstract Ratio subtraction and isosbestic point methods are two innovative spectrophotometric Metronidazole; methods for determining the concentrations of metronidazole (I) and diloxanide furoate (II) in a Diloxanide furoate; mixture. Metronidazole was determined by direct spectrophotometric method at kmax 314.0 nm in Binary mixture; the presence of diloxanide furoate in the range of 4–24 lgml1 with a mean recovery percentage Isosbestic point; of 99.83 ± 1.41. Two spectrophotometric methods were developed for the spectral resolution of Ratio subtraction diloxanide furoate when present in mixture with metronidazole without preliminary separation. The ﬁrst method depends on measuring the absorbance at the isosbestic point at 277.2 nm in the range of 5–30 lgml1 with a mean recovery percentage of 99.96 ± 1.47 for diloxanide furoate. The second method is the ratio subtraction spectroscopic method for spectral isolation of diloxanide furoate present in the mixture which can be measured at 251.2 nm in the range of 5–30 lgml1 with a mean recovery percentage of 99.73 ± 1.33 for diloxanide furoate determination. The suggested procedures were validated using laboratory-prepared mixtures and were successfully applied for the analysis of pharmaceutical preparations. The methods retained their accuracy and precision when the standard addition technique was applied. The results obtained by applying the proposed methods were statistically analyzed and compared with those obtained by the reported method. ª 2010 Cairo University. Production and hosting by Elsevier B.V. All rights reserved.

* Corresponding author. Tel.: +202 33020604. E-mail address: [email protected] (M.R. El-Ghobashy). Introduction

2090-1232 ª 2010 Cairo University. Production and hosting by Metronidazole and diloxanide furoate are formulated together Elsevier B.V. All rights reserved. to be highly effective in the treatment of intestinal and extrain- Peer review under responsibility of Cairo University. testinal amoebic infections. Metronidazole is less effective doi:10.1016/j.jare.2010.06.001 against parasites in the bowel lumen and is, therefore, used in combination with a luminal amoebicide, such as diloxanide furoate in the treatment of invasive amoebiasis. Production and hosting by Elsevier Metronidazole 2-methyl-5-nitroimidazole-1-ethanol [1] was determined individually by short-wave length NIR 324 M.R. El-Ghobashy and N.F. Abo-Talib spectroscopy [2], voltammetry [3–5], NMR spectrometry [6], Chemicals and reagents gas chromatography [7] and HPLC methods either alone [8–10] or in the presence of its metabolites [11,12] or in the All chemicals were of analytical grade and the solvents were of presence of its degradation product [13], in addition to its mix- spectroscopic grade. Methanol, (E-Merck, Darmstadt, ture with other drugs [14,15]. First derivative spectrophotom- Germany). etry was used for the determination of metronidazole in mixture with ciprofloxacin [16]. Standard solutions Diloxanide furoate 2,2-dichloro-N-(4-hydroxyphenyl)- N-methylacetamide [1] was determined individually by colori- Stock solutions metric method [17], in the presence of its degradation product Metronidazole (I) and diloxanide furoate (II) stock solutions by derivative technique, derivative ratio, TLC-densitometry 1 [18] and HPLC [18,19] and in mixture with tinidazole and fura- (1 mg ml ) were prepared by weighing accurately 100 mg of zolidone by second derivative spectrophotometry [20]. each powder into two separate 100 ml volumetric flasks. Meth- The main problem of spectrophotometric binary mixture anol (50 ml) was added, shaken for a few minutes and com- analysis is the simultaneous determination of the two completed to volume with the same solvent. pounds in the same mixture without prior separation. One spectrophotometric determination method has been used for Working solutions resolving such mixture with overlapping spectra, derivative Four micro litres of the stock solution of (I) and 5 ml of the spectrophotometry [21] and HPLC [22]. stock solution (II) were accurately transferred into two sepa- The aim of this work is to develop new spectrophotometric rate 50 ml measuring flasks and diluted to the mark with meth- methods for resolving this mixture with spectral interfering anol to get a final concentration of 80 lgml1 and 100 lgml1 problems, without preliminary separation. The new methods of (I) and (II), respectively. were very simple, did not require any computer programs (derivative and derivative ratio) as metronidazole was deter- Laboratory-prepared mixtures mined by direct spectrophotometry and diloxanide furoate was determined by simple mathematical calculation. Also the Accurate aliquots equivalent to (40–100 lg) of (I) were trans- method used did not require any sophisticated instrumentation, ferred from its working solution (80 lgml1) into a series of such as HPLC, which requires solvents and time. 10 ml volumetric flasks and portions equivalent to (50– 150 lg) of (II) from its working solution (100 lgml1) were Experimental added to the same flasks and volumes were completed to mark with methanol and mixed well. Apparatus Procedures Spectrophotometer: SHIMADZU UV-1601 PC, dual beam UV–visible spectrophotometer with two matched 1 cm quartz Isosbestic spectrophotometric method cells, connected to an IBM compatible personal computer Linearity: aliquots from (I) and (II) working solutions (80 lgml (PC) and an HP-600 inkjet printer. Bundled UV-PC personal 1 of (I) and 100 lgml1 of (II), respectively) equivalent to 40– spectroscopy software version (3.7) was used to process the 240 lg of (I) and 50–300 lg of (II) were transferred into two sep- absorption and the derivative spectra. The spectral band width arate sets of 10 ml volumetric flasks and completed to the mark was 0.2 nm with wavelength scanning speed of 2800 nm min1. with methanol. The zero order absorption spectra were re- corded for both drugs using methanol as a blank; then the absorbance was measured at 314.0 nm for (I) and 277.2 nm Materials (Aiso) for (I) and (II). Two calibration curves were constructed for each drug relating the absorbance at the selected wavelength Pure samples to the corresponding drug concentrations and the regression equations were computed. Metronidazole and diloxanide furoate were kindly supplied by Assay of laboratory-prepared mixtures: Absorbance of the Egyptian Int. Pharmaceutical Industries Co., E.I.P.I.CO. 10th spectra of laboratory-prepared mixtures containing different of Ramadan City, Area B1 P.O. 149, Egypt. Their purity was ratios of (I) and (II) were measured at 314.0 nm corresponding found to be 99.84 ± 1.26 and 100.50 ± 0.71, respectively, to the contents of (I) only, and at 277.2 nm (Aiso) correspond- according to the manufacturer’s direct spectrophotometric ing to the total content of (I) and (II) in the mixture. The con- method (personal communication). centration of (I) alone and the total concentration of the two drugs were calculated from their corresponding regression Market samples equations; then by subtraction of (I) concentration from the total mixture concentration, yielding the actual concentration Furazole tablets (E.I.P.I.CO.); batch no. 080435. It was labeled of (II) in the mixture. to contain 200 and 250 mg metronidazole and diloxanide furoate, respectively, per tablet. Ratio subtraction spectrophotometric method Furazole suspension (E.I.P.I.CO.); batch no. 074135. It was Linearity: Aliquots containing 50–300 lg from (II) working labeled to contain 200 and 100 mg metronidazole and diloxa- solution (80 lgml1) were transferred into a series of 10 ml nide furoate, respectively, per 5 ml. volumetric flasks then completed to volume with methanol; Analysis of metronidazole and diloxanide furoate in binary mixture 325 the spectra of the prepared standard solutions were scanned. A a 100 ml beaker, sonicated in 20 ml methanol for 10 min and calibration curve was constructed relating the absorbance of filtered into a 100 ml volumetric flask. The residue was washed zero order spectra of (II) at kmax 251.2 nm to the correspond- three times using 20 ml methanol each time and the volume ing concentrations and the regression equation was computed. was completed to the mark with methanol. Aliquots of 0.5, 1 Aliquot equivalent to 40 lg from the (I) working solution and 1.5 ml were separately transferred to 10 ml volumetric (100 lgml1) was transferred into 10 ml volumetric flask and flask and diluted with methanol. The general procedures under completed to volume with methanol to be used as a divisor linearity were followed. (4 lgml1). The validity of the methods was assessed by applying the Assay of laboratory-prepared mixtures: Absorbance of the standard addition technique. spectra of laboratory-prepared mixtures containing different Furazole suspension: Furazole (0.5 ml) suspension was accu- ratios of (I) and (II) was scanned; then the (absorbance at each rately transferred into a 100 ml beaker, sonicated in 20 ml wavelength) was divided by the spectrum of 4 lgml1of stan- methanol for 10 min and filtered into a 100 ml volumetric flask. dard (I) (divisor) to obtain division spectra and the absorbance The residue was washed using 20 ml methanol and the volume in the plateau region (the constant) was subtracted. By multi- was completed to the mark with methanol. Aliquots of 0.2, 0.4 plication of the obtained spectra (absorbance at each wave- and 0.6 ml (for analysis of metronidazole) and 0.5, 1 and 1.5 ml length) by the spectrum of the divisor the original curves for (for analysis of diloxanide furoate) were separately transferred direct determination of (II) at 251.2 nm were obtained and to 10 ml volumetric flasks and diluted with methanol. The gen- the concentration was calculated from the corresponding eral procedures under linearity were followed. regression equation. The validity of the methods was assessed by applying the standard addition technique. Assay of pharmaceutical formulations Results and discussion Furazole tablets: Ten Furazole tablets were accurately weighed and finely powdered. A portion equivalent to 8 mg of (I) and Analytical methods for the determination of binary mixture 10 mg of (II) was weighed. The powder was transferred into without previous separation were of interest. Metronidazole

Fig. 1 Zero order absorption spectra of 20 lgml1 of metronidazole (_____), 20 lgml1 of diloxanide furoate (- - - - -) and (1:1) mixture containing 10 lgml1 of each (...... ) using methanol as a blank.

Table 1 Method validation for the determination of pure sample of metronidazole and diloxanide furoate by the proposed methods. Parameter Isosbestic spectrophotometry Ratio subtraction spectrophotometry Metronidazole Diloxanide furoate Diloxanide furoate Accuracy mean ± SD 99.83 ± 1.41 99.96 ± 1.47 99.73 ± 1.33 Precision repeatabilitya 100.07 ± 0.76 99.93 ± 0.48 99.87 ± 0.52 Intermediate precisionb 99.95 ± 0.82 100.05 ± 0.60 100.11 ± 0.59 Concentration range (lgml1) 4–24 5–30 5–30 a The intraday (n = 3), average of three concentrations (4, 12, 24 lgml1) for metronidazole and (5, 15, 25 lgml1) for diloxanide furoate repeated three times within the day. b The interday (n = 3), average of three concentrations (4, 12, 24 lgml1) for metronidazole and (5, 15, 25 lgml1) for diloxanide furoate repeated three times in three successive days. 326 M.R. El-Ghobashy and N.F. Abo-Talib can be determined by direct measurement of absorbance at in the mixture could be calculated, without any interference, at 314.0 nm, while the absorption spectra of diloxanide furoate 314.0 nm. Thus the concentration of (II) could be calculated by and metronidazole showed severe overlap, which makes the subtraction. determination of diloxanide furoate concentration in the mix- A linear correlation was obtained between the absorbance ture more difﬁcult (Fig. 1). By applying the proposed tech- values and the corresponding concentrations of both drugs at niques to the spectral data of the mixture, both diloxanide their corresponding wavelengths. The regression equations furoate and metronidazole concentrations could be determined were: without any interference. Aiso ¼ 0:0134C þ 0:0204 r ¼ 0:9994 at 277:2nm Isosbestic spectrophotometric method A ¼ 0:0324C þ 0:0024 r ¼ 0:9995 at 314:0nm where A is the absorbance, C is the concentration of the drug Erram and Tipnis [23] developed the isosbestic spectrophoto- in lgml1 and r is the correlation coefﬁcient. metric method. This method was used for simultaneous deter- The proposed method was applied for the determination of mination of (I) and (II) in their binary mixtures. At the (I) and (II) in bulk powder: satisfactory results were obtained isosbestic point the mixture of drugs acts as a single compo- (Table 1). nent and gives the same absorbance value as pure drug. Thus, The laboratory-prepared mixtures were analyzed by by measuring the absorbance value at the chosen isosbestic the isosbestic method. The method is valid for determining point 277.2 nm (Aiso)(Fig. 1), the total concentration of both the drug in laboratory-prepared mixtures as shown in (I) and (II) could be calculated, while the concentration of (I) (Table 2).

Table 2 Determination of metronidazole and diloxanide furoate in laboratory-prepared mixtures by the proposed methods. Mixture no. Claimed taken (lgml1) Isospestic spectrophotometry Ratio subtraction spectrophotometry Metronidazole Diloxanide Metronidazole Diloxanide furoate Diloxanide furoate furoate Recoverya% Recoverya%at Recoverya% at 251.2 nm at 314 nm 277.2 nm 1 10 8 101.61 99.00 99.08 2 10 10 98.71 97.26 100.52 3 10 5 101.57 100.86 99.35 4 5 8 97.30 97.45 98.95 5 15 8 100.77 100.29 100.26 6 5 10 98.18 97.07 99.47 Mean ± SD 99.69 ± 1.86 98.66 ± 1.65 99.61 ± 0.64 a Average of three determinations.

Fig. 2 Division spectra of laboratory prepared mixtures of diloxanide furoate (X) and metronidazole (Y) using 4 lgml1 of metronidazole (Y’) as a divisor and methanol as a blank. Analysis of metronidazole and diloxanide furoate in binary mixture 327

Ratio subtraction spectrophotometric method concentration from the corresponding regression equation could be calculated. This can be summarized as follows: The method was applied for determination of mixture of (II) ðII þ IÞ=I0 ¼ II=I0 þ I=I0 ¼ II=I0 þ constant and (I) when the spectrum of (I) extended than the other II=I0 þ constant constant ¼ II=I0 (II), as shown in (Fig. 1). The determination of (II) could be 0 0 achieved by scanning the zero order absorption spectra of II=I I ¼ II the laboratory-prepared mixtures (I and II) in methanol, then The constant can be determined directly from the curve 1 dividing them by a carefully chosen concentration (4 lgml ) (II + I)/I0 by the straight line which is parallel to the wave- of standard (I) (I0 = divisor) to produce a new ratio spectra length axis in the region where (I) is extended. The correct that represents II/I0 + constant, as shown in (Fig. 2); then, choice of the divisor is fundamental, as, if the concentration subtraction of the absorbance values of these constants (I/I0) of the divisor increases or decreases, the resulting constant va- in plateau as shown in (Fig. 3) followed by multiplication of lue will be proportionally decreased or increased [24]. the obtained spectra by (I0) the divisor as shown in (Fig. 4); ﬁ- A linear correlation was obtained between the absorbance nally, the original spectra of (II), which are used for direct and the corresponding concentration of (II) at its correspond- determination of (II) at 251.2 nm, could be obtained and the ing wavelength: the regression equation was:

Fig. 3 Division spectra of laboratory prepared mixtures of diloxanide furoate (X) and metronidazole (Y) using 4 lgml1 of metronidazole (Y0) as a divisor and methanol as a blank after subtraction of the constant.

Fig. 4 The zero order absorption spectra of diloxanide furoate obtained by the proposed ratio subtraction method for the analysis of laboratory prepared mixtures after multiplication by the divisor (Y0). 328 M.R. El-Ghobashy and N.F. Abo-Talib

Table 3 Determination of metronidazole and diloxanide furoate in their pharmaceutical preparations by the proposed methods and application of standard addition technique. Product Isosbestic spectrophotometry Ratio subtraction spectrophotometry Founda (%) ± SD Added Founda Recovery (%) Founda (%) ± SD Added Founda Recovery (%) Metronidazole in Furazole tablets 4 3.94 98.50 4 3.94 98.50 (batch no. 080435) 100.23 ± 0.62 8 8.03 100.38 100.23 ± 0.62 8 8.03 100.38 12 11.77 98.08 12 11.77 98.08 Mean ± SD 98.99 ± 1.23 98.99 ± 1.23 Diloxanide furoate in Furazole tablets 5 4.98 99.60 5 4.96 99.20 (batch no. 080435) 99.04 ± 0.98 10 9.81 98.10 98.97 ± 0.23 10 9.86 98.60 15 14.68 97.87 15 14.80 98.67 Mean ± SD 98.52 ± 0.94 98.82 ± 0.33 Metronidazole in Furazole suspension 4 3.95 98.75 4 3.95 98.75 (batch no. 074135) 98.69 ± 0.54 8 7.87 98.38 98.69 ± 0.54 8 7.87 98.38 12 11.90 99.17 12 11.90 99.17 Mean ± SD 98.77 ± 0.40 98.77 ± 0.40 Diloxanide furoate in Furazole 5 4.98 99.60 5 4.97 99.40 suspension (batch no. 074135) 99.20 ± 0.66 10 10.01 100.10 99.13 ± 0.49 10 9.96 99.60 15 14.72 98.13 15 15.08 100.53 Mean ± SD 99.28 ± 1.02 99.84 ± 0.60 a Average of three determinations.

Table 4 Statistical comparison of the results obtained by applying the proposed methods and the direct spectrophotometric manufacturer method for the analysis of pure metronidazole and diloxanide furoate. Manufacturer method Ratio subtraction spectrophotometry Isosbestic spectrophotometry Value Diloxanide furoate Metronidazole Diloxanide furoate Diloxanide furoate Metronidazole 100.50 99.84 99.73 99.96 99.83 Mean 0.71 1.26 1.33 1.47 1.41 SD 666 66n 0.504 1.588 1.769 2.161 1.988 Variance – – 1.251 0.816 0.013 t-Test (2.228)* – – 3.510 4.288 1.252 F value (5.05)* * The values in parenthesis are corresponding to the theoretical values of t and F (P = 0.05).

A ¼ 0:0726C þ 0:0452 r ¼ 0:9997 between the proposed methods and the manufacturer’s where A is the absorbance of (II) at 251.2 nm, C is the concen- procedure. tration of (II) in lgml1 and r is the correlation coefﬁcient. The proposed method was applied for the determination of Conclusion (II) in bulk powder and satisfactory results were obtained see (Table 1). The aim of this work is to develop simple and new methods for The laboratory-prepared mixtures were analyzed by the ra- the simultaneous determination of metronidazole and diloxa- tio subtraction method at 251.2 nm. The method is valid for nide furoate. The isosbestic spectrophotometric and ratio sub- determining the drug in laboratory-prepared mixtures as traction spectrophotometric methods could be applied to the shown in (Table 2). simultaneous determination of metronidazole and diloxanide The proposed methods were successfully applied for the furoate either in their pure powder form or in their combined analysis of both drugs in pharmaceutical dosage form and preparations. The results demonstrate the usefulness of the the results are shown in (Table 3). methods, which are simple, safe, sensitive, precise, accurate, The validity of the proposed methods was assessed by inexpensive and non-polluting. So, the proposed methods applying the standard addition technique. The results obtained could be used in routine and quality control analysis of metro- were reproducible with low relative standard deviation as nidazole and diloxanide furoate in pharmaceutical prepara- shown in (Table 3). tions containing them. A statistical comparison of the results obtained by the two proposed methods and the manufacturer’s method for pure References drugs is shown in Table 4. The values of the calculated t and F are less than the tabulated ones, which reveals that there is [1] O’Neil MJ, Smith A, Heckelman PE, Budavari SB. Merck index. no signiﬁcant difference with respect to accuracy and precision 14th ed. Darmstadt: Merck Sharp & Dohme Corp.; 2006. Analysis of metronidazole and diloxanide furoate in binary mixture 329

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