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 b
a,*
, Nisreen F. Abo-Talib
b
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt 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 Metronidazole; Diloxanide furoate; Binary mixture; Isosbestic point; Ratio subtraction
Abstract Ratio subtraction and isosbestic point methods are two innovative spectrophotometric methods for determining the concentrations of metronidazole (I) and diloxanide furoate (II) in a mixture. Metronidazole was determined by direct spectrophotometric method at kmax 314.0 nm in the presence of diloxanide furoate in the range of 4–24 lg ml1 with a mean recovery percentage of 99.83 ± 1.41. Two spectrophotometric methods were developed for the spectral resolution of 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 lg ml1 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 lg ml1 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). 2090-1232 ª 2010 Cairo University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of Cairo University. doi:10.1016/j.jare.2010.06.001
Production and hosting by Elsevier
Introduction Metronidazole and diloxanide furoate are formulated together to be highly effective in the treatment of intestinal and extraintestinal amoebic infections. Metronidazole is less effective 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. Metronidazole 2-methyl-5-nitroimidazole-1-ethanol [1] was determined individually by short-wave length NIR
324 spectroscopy [2], voltammetry [3–5], NMR spectrometry [6], gas chromatography [7] and HPLC methods either alone [8–10] or in the presence of its metabolites [11,12] or in the presence of its degradation product [13], in addition to its mixture with other drugs [14,15]. First derivative spectrophotometry was used for the determination of metronidazole in mixture with ciprofloxacin [16]. Diloxanide furoate 2,2-dichloro-N-(4-hydroxyphenyl)N-methylacetamide [1] was determined individually by colorimetric method [17], in the presence of its degradation product by derivative technique, derivative ratio, TLC-densitometry [18] and HPLC [18,19] and in mixture with tinidazole and furazolidone by second derivative spectrophotometry [20]. The main problem of spectrophotometric binary mixture analysis is the simultaneous determination of the two compounds in the same mixture without prior separation. One spectrophotometric determination method has been used for resolving such mixture with overlapping spectra, derivative spectrophotometry [21] and HPLC [22]. The aim of this work is to develop new spectrophotometric methods for resolving this mixture with spectral interfering problems, without preliminary separation. The new methods were very simple, did not require any computer programs (derivative and derivative ratio) as metronidazole was determined by direct spectrophotometry and diloxanide furoate was determined by simple mathematical calculation. Also the method used did not require any sophisticated instrumentation, such as HPLC, which requires solvents and time. Experimental
M.R. El-Ghobashy and N.F. Abo-Talib Chemicals and reagents All chemicals were of analytical grade and the solvents were of spectroscopic grade. Methanol, (E-Merck, Darmstadt, Germany). Standard solutions Stock solutions Metronidazole (I) and diloxanide furoate (II) stock solutions (1 mg ml1) were prepared by weighing accurately 100 mg of each powder into two separate 100 ml volumetric flasks. Methanol (50 ml) was added, shaken for a few minutes and completed to volume with the same solvent. Working solutions Four micro litres of the stock solution of (I) and 5 ml of the stock solution (II) were accurately transferred into two separate 50 ml measuring flasks and diluted to the mark with methanol to get a final concentration of 80 lg ml1 and 100 lg ml1 of (I) and (II), respectively. Laboratory-prepared mixtures Accurate aliquots equivalent to (40–100 lg) of (I) were transferred from its working solution (80 lg ml1) into a series of 10 ml volumetric flasks and portions equivalent to (50– 150 lg) of (II) from its working solution (100 lg ml1) were 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 cells, connected to an IBM compatible personal computer (PC) and an HP-600 inkjet printer. Bundled UV-PC personal spectroscopy software version (3.7) was used to process the absorption and the derivative spectra. The spectral band width was 0.2 nm with wavelength scanning speed of 2800 nm min1. Materials Pure samples Metronidazole and diloxanide furoate were kindly supplied by Egyptian Int. Pharmaceutical Industries Co., E.I.P.I.CO. 10th of Ramadan City, Area B1 P.O. 149, Egypt. Their purity was found to be 99.84 ± 1.26 and 100.50 ± 0.71, respectively, according to the manufacturer’s direct spectrophotometric method (personal communication). Market samples Furazole tablets (E.I.P.I.CO.); batch no. 080435. It was labeled to contain 200 and 250 mg metronidazole and diloxanide furoate, respectively, per tablet. Furazole suspension (E.I.P.I.CO.); batch no. 074135. It was labeled to contain 200 and 100 mg metronidazole and diloxanide furoate, respectively, per 5 ml.
Isosbestic spectrophotometric method Linearity: aliquots from (I) and (II) working solutions (80 lg ml 1 of (I) and 100 lg ml1 of (II), respectively) equivalent to 40– 240 lg of (I) and 50–300 lg of (II) were transferred into two separate sets of 10 ml volumetric flasks and completed to the mark with methanol. The zero order absorption spectra were recorded for both drugs using methanol as a blank; then the absorbance was measured at 314.0 nm for (I) and 277.2 nm (Aiso) for (I) and (II). Two calibration curves were constructed for each drug relating the absorbance at the selected wavelength to the corresponding drug concentrations and the regression equations were computed. Assay of laboratory-prepared mixtures: Absorbance of the spectra of laboratory-prepared mixtures containing different ratios of (I) and (II) were measured at 314.0 nm corresponding to the contents of (I) only, and at 277.2 nm (Aiso) corresponding to the total content of (I) and (II) in the mixture. The concentration of (I) alone and the total concentration of the two drugs were calculated from their corresponding regression equations; then by subtraction of (I) concentration from the total mixture concentration, yielding the actual concentration of (II) in the mixture. Ratio subtraction spectrophotometric method Linearity: Aliquots containing 50–300 lg from (II) working solution (80 lg ml1) were transferred into a series of 10 ml volumetric flasks then completed to volume with methanol;
Analysis of metronidazole and diloxanide furoate in binary mixture
325
a 100 ml beaker, sonicated in 20 ml methanol for 10 min and filtered into a 100 ml volumetric flask. The residue was washed three times using 20 ml methanol each time and the volume was completed to the mark with methanol. Aliquots of 0.5, 1 and 1.5 ml were separately transferred to 10 ml volumetric flask and diluted with methanol. The general procedures under linearity were followed. The validity of the methods was assessed by applying the standard addition technique. Furazole suspension: Furazole (0.5 ml) suspension was accurately transferred into a 100 ml beaker, sonicated in 20 ml methanol for 10 min and filtered into a 100 ml volumetric flask. The residue was washed using 20 ml methanol and the volume was completed to the mark with methanol. Aliquots of 0.2, 0.4 and 0.6 ml (for analysis of metronidazole) and 0.5, 1 and 1.5 ml (for analysis of diloxanide furoate) were separately transferred to 10 ml volumetric flasks and diluted with methanol. The general procedures under linearity were followed. The validity of the methods was assessed by applying the standard addition technique.
the spectra of the prepared standard solutions were scanned. A calibration curve was constructed relating the absorbance of zero order spectra of (II) at kmax 251.2 nm to the corresponding concentrations and the regression equation was computed. Aliquot equivalent to 40 lg from the (I) working solution (100 lg ml1) was transferred into 10 ml volumetric flask and completed to volume with methanol to be used as a divisor (4 lg ml1). Assay of laboratory-prepared mixtures: Absorbance of the spectra of laboratory-prepared mixtures containing different ratios of (I) and (II) was scanned; then the (absorbance at each wavelength) was divided by the spectrum of 4 lg ml1of standard (I) (divisor) to obtain division spectra and the absorbance in the plateau region (the constant) was subtracted. By multiplication of the obtained spectra (absorbance at each wavelength) by the spectrum of the divisor the original curves for direct determination of (II) at 251.2 nm were obtained and the concentration was calculated from the corresponding regression equation. 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 10 mg of (II) was weighed. The powder was transferred into
Analytical methods for the determination of binary mixture without previous separation were of interest. Metronidazole
Fig. 1 Zero order absorption spectra of 20 lg ml1 of metronidazole (_____), 20 lg ml1 of diloxanide furoate (- - - - -) and (1:1) mixture containing 10 lg ml1 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
Accuracy mean ± SD Precision repeatabilitya Intermediate precisionb Concentration range (lg ml1)
Isosbestic spectrophotometry
Ratio subtraction spectrophotometry
Metronidazole
Diloxanide furoate
Diloxanide furoate
99.83 ± 1.41 100.07 ± 0.76 99.95 ± 0.82 4–24
99.96 ± 1.47 99.93 ± 0.48 100.05 ± 0.60 5–30
99.73 ± 1.33 99.87 ± 0.52 100.11 ± 0.59 5–30
a The intraday (n = 3), average of three concentrations (4, 12, 24 lg ml1) for metronidazole and (5, 15, 25 lg ml1) for diloxanide furoate repeated three times within the day. b The interday (n = 3), average of three concentrations (4, 12, 24 lg ml1) for metronidazole and (5, 15, 25 lg ml1) for diloxanide furoate repeated three times in three successive days.
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M.R. El-Ghobashy and N.F. Abo-Talib
can be determined by direct measurement of absorbance at 314.0 nm, while the absorption spectra of diloxanide furoate and metronidazole showed severe overlap, which makes the determination of diloxanide furoate concentration in the mixture more difficult (Fig. 1). By applying the proposed techniques to the spectral data of the mixture, both diloxanide furoate and metronidazole concentrations could be determined without any interference.
in the mixture could be calculated, without any interference, at 314.0 nm. Thus the concentration of (II) could be calculated by subtraction. A linear correlation was obtained between the absorbance values and the corresponding concentrations of both drugs at their corresponding wavelengths. The regression equations were:
Isosbestic spectrophotometric method
Aiso ¼ 0:0134C þ 0:0204 r ¼ 0:9994 at 277:2 nm A ¼ 0:0324C þ 0:0024 r ¼ 0:9995 at 314:0 nm
Erram and Tipnis [23] developed the isosbestic spectrophotometric method. This method was used for simultaneous determination of (I) and (II) in their binary mixtures. At the isosbestic point the mixture of drugs acts as a single component and gives the same absorbance value as pure drug. Thus, by measuring the absorbance value at the chosen isosbestic point 277.2 nm (Aiso) (Fig. 1), the total concentration of both (I) and (II) could be calculated, while the concentration of (I)
where A is the absorbance, C is the concentration of the drug in lg ml1 and r is the correlation coefficient. The proposed method was applied for the determination of (I) and (II) in bulk powder: satisfactory results were obtained (Table 1). The laboratory-prepared mixtures were analyzed by the isosbestic method. The method is valid for determining the drug in laboratory-prepared mixtures as shown in (Table 2).
Table 2
Determination of metronidazole and diloxanide furoate in laboratory-prepared mixtures by the proposed methods.
Mixture no.
1 2 3 4 5 6
Claimed taken (lg ml1)
Isospestic spectrophotometry
Ratio subtraction spectrophotometry
Metronidazole
Metronidazole
Diloxanide furoate
Diloxanide furoate
Recoverya% at 314 nm
Recoverya% at 277.2 nm
Recoverya% at 251.2 nm
101.61 98.71 101.57 97.30 100.77 98.18
99.00 97.26 100.86 97.45 100.29 97.07
99.08 100.52 99.35 98.95 100.26 99.47
10 10 10 5 15 5
Mean ± SD a
Diloxanide furoate
8 10 5 8 8 10
99.69 ± 1.86
98.66 ± 1.65
99.61 ± 0.64
Average of three determinations.
Fig. 2 Division spectra of laboratory prepared mixtures of diloxanide furoate (X) and metronidazole (Y) using 4 lg ml1 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) and (I) when the spectrum of (I) extended than the other (II), as shown in (Fig. 1). The determination of (II) could be achieved by scanning the zero order absorption spectra of the laboratory-prepared mixtures (I and II) in methanol, then dividing them by a carefully chosen concentration (4 lg ml1) of standard (I) (I0 = divisor) to produce a new ratio spectra that represents II/I0 + constant, as shown in (Fig. 2); then, subtraction of the absorbance values of these constants (I/I0 ) in plateau as shown in (Fig. 3) followed by multiplication of the obtained spectra by (I0 ) the divisor as shown in (Fig. 4); finally, the original spectra of (II), which are used for direct determination of (II) at 251.2 nm, could be obtained and the
ðII þ IÞ=I0 ¼ II=I0 þ I=I0 ¼ II=I0 þ constant II=I0 þ constant constant ¼ II=I0 II=I0 I0 ¼ II The constant can be determined directly from the curve (II + I)/I0 by the straight line which is parallel to the wavelength axis in the region where (I) is extended. The correct choice of the divisor is fundamental, as, if the concentration of the divisor increases or decreases, the resulting constant value will be proportionally decreased or increased [24]. A linear correlation was obtained between the absorbance and the corresponding concentration of (II) at its corresponding wavelength: the regression equation was:
Fig. 3 Division spectra of laboratory prepared mixtures of diloxanide furoate (X) and metronidazole (Y) using 4 lg ml1 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 ).
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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 (batch no. 080435)
100.23 ± 0.62
4 8 12
3.94 8.03 11.77
98.50 100.38 100.23 ± 0.62 98.08 98.99 ± 1.23
4 8 12
3.94 8.03 11.77
98.50 100.38 98.08 98.99 ± 1.23
5 10 15
4.98 9.81 14.68
99.60 98.10 98.97 ± 0.23 97.87 98.52 ± 0.94
5 10 15
4.96 9.86 14.80
99.20 98.60 98.67 98.82 ± 0.33
4 8 12
3.95 7.87 11.90
98.75 98.38 98.69 ± 0.54 99.17 98.77 ± 0.40
4 8 12
3.95 7.87 11.90
98.75 98.38 99.17 98.77 ± 0.40
5 10 15
4.98 10.01 14.72
99.60 100.10 99.13 ± 0.49 98.13 99.28 ± 1.02
5 10 15
4.97 9.96 15.08
99.40 99.60 100.53 99.84 ± 0.60
Mean ± SD Diloxanide furoate in Furazole tablets (batch no. 080435) 99.04 ± 0.98 Mean ± SD Metronidazole in Furazole suspension (batch no. 074135) 98.69 ± 0.54 Mean ± SD Diloxanide furoate in Furazole suspension (batch no. 074135)
99.20 ± 0.66
Mean ± SD 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
Diloxanide furoate
Metronidazole
Diloxanide furoate
Diloxanide furoate
Metronidazole
100.50 0.71 6 0.504 – –
99.84 1.26 6 1.588 – –
99.73 1.33 6 1.769 1.251 3.510
99.96 1.47 6 2.161 0.816 4.288
99.83 1.41 6 1.988 0.013 1.252
*
Value
Mean SD n Variance t-Test (2.228)* 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 where A is the absorbance of (II) at 251.2 nm, C is the concentration of (II) in lg ml1 and r is the correlation coefficient. The proposed method was applied for the determination of (II) in bulk powder and satisfactory results were obtained see (Table 1). The laboratory-prepared mixtures were analyzed by the ratio subtraction method at 251.2 nm. The method is valid for determining the drug in laboratory-prepared mixtures as shown in (Table 2). The proposed methods were successfully applied for the analysis of both drugs in pharmaceutical dosage form and the results are shown in (Table 3). The validity of the proposed methods was assessed by applying the standard addition technique. The results obtained were reproducible with low relative standard deviation as shown in (Table 3). A statistical comparison of the results obtained by the two proposed methods and the manufacturer’s method for pure 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 no significant difference with respect to accuracy and precision
between the proposed methods and the manufacturer’s procedure. Conclusion The aim of this work is to develop simple and new methods for the simultaneous determination of metronidazole and diloxanide furoate. The isosbestic spectrophotometric and ratio subtraction spectrophotometric methods could be applied to the simultaneous determination of metronidazole and diloxanide furoate either in their pure powder form or in their combined preparations. The results demonstrate the usefulness of the methods, which are simple, safe, sensitive, precise, accurate, inexpensive and non-polluting. So, the proposed methods could be used in routine and quality control analysis of metronidazole and diloxanide furoate in pharmaceutical preparations containing them. References [1] O’Neil MJ, Smith A, Heckelman PE, Budavari SB. Merck index. 14th ed. Darmstadt: Merck Sharp & Dohme Corp.; 2006.
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