Analytical Letters, 42: 15711587, 2009 Copyright # Taylor & Francis Group, LLC ISSN: 0003-2719 print=1532-236X online

DOI: 10.1080/00032710902993795

PHARMACEUTICAL ANALYSIS

Spectrophotometric Determination of Risedronate and Etidronate in Pharmaceutical Formulations via the Molybdovanadate Method
Mohamed I. Walash, Mohamed E.-S. Metwally, Manal I. Eid, and Rania N. El-Shaheny
Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura, Egypt

Abstract: An accurate and sensitive spectrophotometric method was developed for the determination of risedronate and etidronate in pharmaceuticals. The method was based on oxidation of the studied drugs with potassium persulfate and reaction of the generated orthophosphate ions with molybdovanadate reagent. The produced yellow phosphovanadomolybdate complex was measured at 313 nm. The method was rectilinear in the ranges 0.510 and 0.58 mg=mL with detection limits of 0.087 and 0.122 mg=mL for risedronate and etidronate, respectively. The method was applied for the determination of the studied drugs in their tablets, and the results agreed with those obtained by the comparison methods. Keywords: Etidronate, molybdovanadate, risedronate, spectrophotometry

INTRODUCTION Risedronate sodium, (I), is sodium trihydrogen [1-hydroxy-2-(3-pyridyl) ethylidene] diphosphonate, and etidronate disodium, (II), is disodium dihydrogen (1-hydroxyethylidene) diphosphonate (Scheme 1). Both
Received 3 December 2008; accepted 15 April 2009. Address correspondence to Manal I. Eid, Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, 35516 Mansoura, Egypt. E-mail: manal_eid@yahoo.com 1571

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Scheme 1. Structures of the studied drugs.

belong to the bisphosphonates, which are analogs of pyrophosphate, in which the central oxygen atom is replaced by a carbon atom with two further substituents. They are given in bone disorders in which excessive bone resorption is a problem, such as Pagets disease of bone and osteoporosis. The clinical utility of bisphosphonates resides in their ability to inhibit bone resorption. The mechanism by which this antiresorptive effect occurs is not completely known, but it is thought that the bisphosphonate becomes incorporated into the bone matrix and is imbibed by osteoclasts during resorption (Martindale 2007; Goodman and Gilman 2001). Compound (I) is not officially available in any pharmacopoeia. A review of the literature revealed that few analytical methods have been reported for its determination. The techniques that are currently used in this connection include spectrophotometry (Taha and Youssef 2003), liquid chromatographytandem mass spectrometry (Zhu et al. 2006), and enzyme-linked immunosorbent assay (Mitchell et al. 2000). Different ion-pair high-performance liquid chromatographic (HPLC) methods are used for determination of (I) in tablets or biological fluids (Jia, Li, and Zhao 2006; Auoch et al. 2004; Kyriakides and Panderi 2007; Vallano et al. 2003). The United States Pharmacopeia (2005) and the British Pharmacopeia (2007) describe different titrimetric methods for the assay of (II). The literature includes many different techniques that are used currently for determination of (II), including titrimetry (Wang et al. 2002; Janecki, Michalowski, and Zielinski 2000; Podolska et al. 1997; Podolska, Bialeka, and Kwiatkowska-Puchniarz 2000), spectrophotometry (Taha and Youssef 2003; Shallan 2007), spectrofluorometry (Jing, Mu, and Ou 1997), gas chromatography (Ismail et al. 1987), ion chromatography (United States Pharmacopoeia 2007; Tsai et al. 1994; Thompson et al. 1994; Tsai, Ip, and Brooks 1993; Fernandes, Leite, and Lancas 2007;

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Forbes et al. 1989; Xie, Jiang, and Zhang, 2006; Nowack 1997), and HPLC (Shallan 2007; Liu et al. 2008). The chromatographic techniques reported for the determination of the two drugs are unattractive for routine analysis because they require sophisticated instrumentation, are expensive, and have a poor sampling rate. Although spectrophotometric and spectrofluorometric methods are generally simple and require low-cost and widely available instrumentation, they are time-consuming or not very sensitive. Moreover, the titrimetric techniques are generally not attractive techniques for quality-control laboratories because of time consumption and lack of sensitivity. The present work describes a simple, sensitive, and reproducible assay for the determination of the two drugs in their dosage forms. The method was based on quantitative thermal-induced oxidation of the studied drugs by heating with potassium persulfate as the oxidizing agent. The produced orthophosphate ions were then determined by the reaction with molybdovanadic acid. The proposed method is of great value in quality control analysis of (I) and (II) because it is simple, rapid, and sensitive and does not require expensive instruments or critical analytical reagents.

EXPERIMENTAL Apparatus A Shimadzu recording spectrophotometer (UV-1601, P=N 206-67001) with 1-cm matched cells was used.

Materials and Reagents All the reagents were analytical grade, and water was always double distilled. Risedronate sodium, 100.41% pure sample (Taha and Youssef 2003), was kindly provided by the Alkan Pharma Co., Cairo, Egypt (batch FRS=001=06-07). Etidronate disodium, with a purity of 98.13% (British Pharmacopeia 2007), was kindly donated by Memphis Co. for Pharmaceutical and Chemical Industries, Cairo, Egypt. Tablets containing risedronate sodium (Actonel tablets, batch 406767, labeled to contain 5 mg of risedronate sodium=tablet, Aventis Pharma Co., Cairo, Egypt) were obtained from commercial sources in a local market. Tablets containing etidronate disodium (Etidron tablets, batch 305741, labeled to contain 200 mg of etidronate disodium=tablet, Memphis Co. for

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Pharmaceutical and Chemical Industries, Cairo, Egypt) were purchased from commercial sources in a local market. Potassium dihydrogen orthophosphate and potassium persulfate were obtained from Winlab (Middlesex, England). Ammonium molybdate and hydrochloric acid (labeled to have purities of 98% and 32%, respectively) were obtained from Merck (Darmstadt, Germany). Ammonium metavanadate (labeled to have a purity of 99%) was purchased from Aldrich Chemical Co. Ltd. (Gillingham, Dorest, England). Molybdovanadate reagent was prepared by mixing 0.5 g ammonium molybdate and 0.025 g ammonium metavanadate, diluting the mixture with distilled water to about 50 mL, and sonicating it in an ultrasonic bath until the solids completely dissolved. The solution initially was intensely yellow. After 7.4 mL conc. HCl was added, the yellow color faded away, and the mixture was diluted to 100 mL with distilled water. The solution was stable for at least 3 months when kept in the refrigerator. All glassware was cleaned effectively with special care to avoid phosphate contamination. All glassware was thoroughly rinsed with hot 1:1 HCl and then rinsed many times with doubly distilled water.

Stock Solutions Standard stock solutions containing 0.82 mM of (I) and 1.00 mM of (II) were prepared by dissolving 25.0 mg of each drug in 100 mL distilled water. These solutions were stable for at least 10 days when stored in the refrigerator and protected from light. More dilute solutions were obtained by appropriate dilution. Standard phosphate solutions containing 1.64 mM and 2.00 mM of potassium dihydrogen orthophosphate were prepared.

General Recommended Procedures Procedure for Calibration Graph Accurately measured aliquots of the stock solutions were transferred into a series of 25-mL volumetric flasks so that the final concentrations were in the range of 0.510 mg=mL for (I) and 0.58 mg=mL for (II). Then 2 and 1 mL of 0.2% potassium persulfate solution for (I) and (II), respectively, were added, and the solutions were mixed well and heated in a boiling water bath for 10 min for (I) and 5 min for (II). The solutions were cooled, and 0.8 mL of molybdovanadate reagent was added for both

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(I) and (II). The mixtures were shaken well and completed to volume with distilled water. The absorbance was measured at 313 nm against an appropriate blank, prepared simultaneously. To get the standard calibration graphs, the values of the absorbance were plotted against the final concentration in mg=mL. Alternatively, the regression equations were derived. Procedure for Determination of the Studied Drugs in Dosage Forms The coatings of the Actonel tablets were removed. Accurately weighed quantities of the mixed contents of 10 pulverized tablets (Actonel and Etidron), equivalent to 25.0 mg of each drug, were transferred into 100-mL volumetric flasks. The solutions were completed to the mark with distilled water. The contents of the flasks were sonicated for 15 min and filtered. The nominal contents were calculated either from the previously plotted calibration graphs or using the corresponding regression equations.

RESULTS AND DISCUSSION The molybdovanadate method plays a prominent role in the determination of inorganic, organic phosphate (Baadenhuljsen, Seuren-Jacobs, and Jansen 1977; Mas-Torres et al. 1997; Lin and Morales 1997; Ueda and Wada 1970; Bartlett and Lewis 1970; Richardson 1964) and some phosphorus-containing drugs viz., alendronate sodium (Zhang et al. 2000) and phosphonoformate (Forsman, Andersson, and Tornros 1986). For determination of organic phosphate and phosphoruscontaining drugs, the method is based on oxidative cleavage of the phosphoruscarbon (PC) bond and the colorimetric determination of the orthophosphate ions produced after its conversion to the yellow phosphovanadomolybdate complex. It should be noted that it is not necessary to achieve complete conversion of the analyte to measure it in pharmaceutical formulations. The studied drugs belong to the bisphosphonate group, and the proposed method depends on their oxidation by heating with potassium persulfate. The generated orthophosphate ions are then determined by conversion to phosphovanadomolybdate complex, which has maximum absorbance at 313 nm (Fig. 1). To study the factors affecting the thermal-induced oxidation of the studied drugs, standard phosphate solutions (1.64 and 2.00 mM solutions) were prepared and used for the determination of the percentage conversion of the studied drugs to orthophosphate ions.

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Figure 1. Absorption spectra of the formed complexes: ( ) etidronate (4 mg=mL); () risedronate (4 mg=mL).

Optimization of Experimental Conditions Factors affecting the thermal-induced oxidation of the drugs as well as the different experimental parameters affecting the color development and stability were carefully studied and optimized.

Effect of Potassium Persulfate Concentration The effect of potassium persulfate concentration was studied, and the experimental results are depicted in Fig. 2. As can be seen, maximum and constant percentage conversions (ca. 75% and 89% for (I) and (II), respectively) were achieved at volumes of 1.5 and 0.5 mL of 0.2% potassium persulfate solution for (I) and (II), respectively, after which further increase of the volume of persulfate has no effect. So, 2 and 1 mL of 0.2% potassium persulfate solution were chosen as optimal for (I) and (II), respectively.

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Figure 2. Effect of volume of 0.2% potassium persulfate on percentage conversion of the studied drugs: (&) risedronate (8 mg=mL); (~) etidronate (5 mg=mL).

Effect of Temperature The effect of heating temperature was studied in the range of 30100 C. No conversion of the analytes was observed at temperatures less than 50 C, whereas maximum quantitative conversion was achieved at temperatures greater than 90 C for both drugs (Fig. 3). A temperature of 100 C was chosen as optimal for both drugs.

Figure 3. Effect of heating temperature on percentage conversion of the studied drugs: (&) risedronate (8 mg=mL); (~) etidronate (5 mg=mL).

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Figure 4. Effect of heating time on percentage conversion of the studied drugs: (&) risedronate (8 mg=mL); (~) etidronate (5 mg=mL).

Effect of Heating Time The effect of heating time on the oxidation of the two drugs was studied. It was found that 7 min and 3 min were sufficient for maximum conversion of (I) and (II) (ca. 75% and 89%), respectively (Fig. 4). So, 10 min and 5 min were selected as optimum heating times for (I) and (II), respectively, to ensure maximum oxidation of the studied drugs. Effect of Molybdovanadate Concentration Maximum color development was achieved with volumes of 0.51.1 mL of the molybdovanadate reagent for both drugs. Larger volumes of the reagent cause a slight decrease of the absorbance (Fig. 5), noisy peaks, and increase of blank readings. So, 0.8 mL was used for all experiments within the concentration ranges to ensure maximum absorbance and minimum blank readings. Effect of Acidity The effect of acidity was studied in the range of 0.41.6 M HCl. At low acidities, yellow-orange molybdenumvanadium complexes were formed and led to unacceptable high-absorbing blanks; at high acidities, the rate of color formation was slow, causing a decrease of the absorbance and nonlinear response. The optimum medium for preparation of the

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Figure 5. Effect of volume of molybdovanadate reagent on the absorbance of the formed complexes: (&) risedronate (8 mg=mL); (~) etidronate (5 mg=mL).

molybdovanadate reagent was 0.75 M HCl because it gave maximum absorbance and minimum blank readings. Effect of Time on Stability of the Product The absorbance of the colored complexes reached the maximum value immediately after the addition of the molybdovanadate reagent and remained constant at room temperature for at least 2 h.

METHOD VALIDATION Concentration Ranges and Calibration Graphs The calibration graphs obtained by plotting the values of the absorbance vs. the final concentrations were rectilinear over the concentration ranges cited in Table 1. Linear regression analysis of the data gave the following equations: Risedronate: A 0:0018 0:1127C Etidronate: A 0:0273 0:1482C r 0:9999 r 0:9999

where A is the absorbance at 313 nm, C is the concentration in mg=mL, and r is the correlation coefficient.

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Table 1. Analytical data for the proposed method Parametera Concentration range (mg=mL) Limit of detection (LOD) (mg=mL) Limit of quantification (LOQ) (mg=mL) Correlation coefficient Slope Intercept Sy=x Sa Sb RSD (%) Error (%) e (l=mol.=cm.) Risedronate 0.510 0.094 0.286 0.9999 0.1127 0.0018 4.09 103 3.22 103 4.59 104 0.96 0.37 3.482 104 Etidronate 0.58 0.134 0.405 0.9999 0.1482 0.0273 6.55 103 6.00 103 9.67 104 1.02 0.46 3.948 104

a Sy=x: standard deviation of the residuals; Sb: standard deviation of p the slope; Error % RSD= n; Sa, standard deviation of the intercept; and E, molar absorbtivity.

Statistical analysis of the data gave small values of the standard deviations of the residuals (Sy=x), the standard deviation of the intercept (Sa), the standard deviation of the slope (Sb), and the percentage of relative error (% Er) as shown in Table 1.

Limit of Quantitation and Limit of Detection The limit of quantitation (LOQ) was determined by establishing the weakest concentration that can be measured according to the ICH Q2(R1) recommendation (ICH 2005), below which the calibration graph is nonlinear; it was found to be 0.286 and 0.405 mg=mL for (I) and (II), respectively. The limit of detection (LOD) was determined by evaluating the weakest concentration of the analytes that can be readily detected and was found to be 0.094 and 0.134 mg=mL (3.081 107 and 5.36 107 M=L) for (I) and (II), respectively. The LOQ and LOD were calculated according to the following equations (ICH 2005): LOQ 10Sa =b LOD 3:3Sa =b

Table 2. Accuracy and precision data for the studied drugs using the proposed method Interday precision Recovery (%) Concentration added (mg=mL) Concentration found (mg=mL) Recovery (%)

Intraday precision

Parameter

Concentration added (mg=mL)

Concentration found (mg=mL)

Risedronate 3.974 5.929 8.030 4.0 6.0 8.0 4.043 5.963 7.930

4.0 6.0 8.0

1581 0.995 4.054 5.968 1.0 4.0 6.0 99.50 101.35 99.47 100.11 1.08 1.08 0.62

x SD RSD (%) Error (%)

99.35 98.81 100.40 99.52 0.81 0.81 0.47 0.9815 4.017 5.968

101.08 99.38 99.14 99.87 1.06 1.06 0.61

Etidronate

1.0 4.0 6.0

x SD % RSD % Er

98.15 100.43 99.47 99.35 1.15 1.16 0.67

Note. Each result is the average of three separate assays.

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where Sa is the standard deviation of the intercept of regression line and b is the slope of the calibration curve.

Accuracy and Precision The intraday precision and accuracy of the assay were measured by analyzing three concentrations in one day. Also, the interday precision and accuracy were determined over three successive days by analyzing the same concentrations. The obtained results for both the intra- and interday precision and accuracy are abridged in Table 2.

Table 3. Application of the proposed and comparison methods to the assay of the studied drugs in active pharmaceutical ingredient and reference forms Proposed method Conc. added (mg=mL) Conc. found (mg=mL) Recovery (%) Comparison method Conc. added (mg=mL) Recovery (%)

Parameter Risedronate

0.5 1.0 2.0 4.0 6.0 8.0 10.0 x SD t F Etidronate 2.0 4.0 5.0 6.0 8.0 x SD t F

0.490 0.995 1.981 4.004 6.060 8.021 9.956

98.00 99.50 99.05 100.10 101.00 100.26 99.56 99.64 0.96 1.201 (2.306) 1.306 (19.33) 99.80 101.95 99.56 100.40 99.48 100.24 1.02 0.401 (2.447) 2.134 (19.25)

4.0 8.0 10.0

99.90 101.38 99.96

100.41 0.84

1.996 4.078 4.978 6.024 7.958

4.0 8.0 10.0

98.98 101.66 99.18

99.94 1.49

Note. Each result is the average of three separate assays. Values between brackets are the tabulated t and F values at p 0.05.

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APPLICATION OF THE PROPOSED METHOD The proposed method was applied to the determination of the studied drugs in active pharmaceutical ingredients, reference forms, and tablets. The percentage recoveries of the studied drugs compared with those obtained by the comparison method (Taha and Youssef 2003) are given in Tables 3 and 4. The comparison method involved the spectrophotometric determination of the two drugs by oxidation with ceric(IV) sulfate in 0.5 M sulfuric acid and subsequent measurement of the excess unreacted ceric(IV) sulfate at 320 nm. Statistical analysis (Miller and Miller 2005) of the results obtained by the proposed and comparison methods using Students t-test and variance ratio F-test revealed no significant differences between the accuracy and precision of the two methods.

Table 4. Application of the proposed and comparison methods to the assay of the studied drugs in dosage forms Proposed method Conc. Conc. found added (mg=mL) (mg=mL) Recovery (%) Comparison method Conc. added (mg=mL) Recovery (%)

Parameter

Actonel tablets (5 mg risedronate 2.0 4.0 6.0 x SD t F

sodium=tablet) 1.972 98.60 3.960 99.00 6.015 100.25 99.28 0.86 1829 (2.776) 2.046 (19.00)

4.0 100.38 8.0 99.95 10.0 102.27 100.87 1.23

Etidron tablets (200 mg etidronate disodium=tablet) 1.0 1.018 101.80 4.0 4.011 100.28 6.0 5.956 99.16 x SD 100.08 0.84 t 0.969 (2.776) F 1.123 (19.00)

4.0 100.60 8.0 99.09 10.0 99.04 99.58 0.89

Note. Each result is the average of three separate assays. Values between brackets are the tabulated t and F values at p 0.05.

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Scheme 2. Proposed scheme of the oxidation reaction of the studied drugs with potassium persulfate under the described reaction conditions.

MECHANISM OF THE REACTION First Step The first step involved oxidation of the studied drugs by heating with potassium persulfate as the oxidizing agent, yielding orthophosphate ions as shown in Scheme 2. Second Step In the second step of the reaction, the generated orthophosphate ions reacted with ammonium molybdate and ammonium metavanadate to yield phosphovanadomolybdate complex, which has a yellow color measured at 313 nm. CONCLUSION A simple and sensitive spectrophotometric method was developed for determination of etidronate and risedronate in either active pharmaceutical ingredients or commercial tablets. Compared to previously reported chromatographic methods for the determination of the studied drugs, our method is simple and inexpensive. Additionally, it does not require the elaborate procedures associated with chromatographic methods. In comparison to other reported spectrophotometric methods currently used for determination of the studied drugs, our method is superior in terms of sensitivity and speed. REFERENCES
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