PHARMACEUTICAL ANALYSIS

Spectrophotometric Determination ofRisedronate and Etidronate in PharmaceuticalFormulations via the Molybdovanadate Method

Mohamed I. Walash, Mohamed E.-S. Metwally, Manal I. Eid,

and Rania N. El-ShahenyDepartment of Analytical Chemistry, Faculty of Pharmacy,

University of Mansoura, Mansoura, Egypt

Abstract: An accurate and sensitive spectrophotometric method was developedfor the determination of risedronate and etidronate in pharmaceuticals. Themethod was based on oxidation of the studied drugs with potassium persulfateand reaction of the generated orthophosphate ions with molybdovanadatereagent. The produced yellow phosphovanadomolybdate complex was measuredat 313 nm. The method was rectilinear in the ranges 0.5–10 and 0.5–8mg=mL withdetection limits of 0.087 and 0.122mg=mL for risedronate and etidronate, respec-tively. The method was applied for the determination of the studied drugs in theirtablets, 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 disodiumdihydrogen (1-hydroxyethylidene) diphosphonate (Scheme 1). Both

Received 3 December 2008; accepted 15 April 2009.Address correspondence to Manal I. Eid, Department of Analytical Chemis-

try, Faculty of Pharmacy, University of Mansoura, 35516 Mansoura, Egypt.E-mail: manal_eid@yahoo.com

Analytical Letters, 42: 1571–1587, 2009Copyright # Taylor & Francis Group, LLCISSN: 0003-2719 print=1532-236X onlineDOI: 10.1080/00032710902993795

1571

belong to the bisphosphonates, which are analogs of pyrophosphate,in which the central oxygen atom is replaced by a carbon atom withtwo further substituents. They are given in bone disorders in which exces-sive bone resorption is a problem, such as Paget’s disease of bone andosteoporosis. The clinical utility of bisphosphonates resides in their abil-ity to inhibit bone resorption. The mechanism by which this antiresorp-tive effect occurs is not completely known, but it is thought that thebisphosphonate becomes incorporated into the bone matrix and isimbibed by osteoclasts during resorption (Martindale 2007; Goodmanand Gilman 2001).

Compound (I) is not officially available in any pharmacopoeia. Areview of the literature revealed that few analytical methods have beenreported for its determination. The techniques that are currently usedin this connection include spectrophotometry (Taha and Youssef 2003),liquid chromatography–tandem mass spectrometry (Zhu et al. 2006),and enzyme-linked immunosorbent assay (Mitchell et al. 2000). Differention-pair high-performance liquid chromatographic (HPLC) methods areused for determination of (I) in tablets or biological fluids (Jia, Li, andZhao 2006; Auoch et al. 2004; Kyriakides and Panderi 2007; Vallanoet al. 2003).

The United States Pharmacopeia (2005) and the British Pharmacopeia(2007) describe different titrimetric methods for the assay of (II). The lit-erature includes many different techniques that are used currently fordetermination of (II), including titrimetry (Wang et al. 2002; Janecki,Michalowski, and Zielinski 2000; Podolska et al. 1997; Podolska, Bia-leka, and Kwiatkowska-Puchniarz 2000), spectrophotometry (Taha andYoussef 2003; Shallan 2007), spectrofluorometry (Jing, Mu, and Ou1997), 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;

Scheme 1. Structures of the studied drugs.

1572 M. I. Walash et al.

Forbes et al. 1989; Xie, Jiang, and Zhang, 2006; Nowack 1997), andHPLC (Shallan 2007; Liu et al. 2008).

The chromatographic techniques reported for the determination ofthe two drugs are unattractive for routine analysis because they requiresophisticated instrumentation, are expensive, and have a poor samplingrate. Although spectrophotometric and spectrofluorometric methodsare generally simple and require low-cost and widely available instru-mentation, they are time-consuming or not very sensitive. Moreover,the titrimetric techniques are generally not attractive techniques forquality-control laboratories because of time consumption and lack ofsensitivity.

The present work describes a simple, sensitive, and reproducibleassay for the determination of the two drugs in their dosage forms.The method was based on quantitative thermal-induced oxidation ofthe studied drugs by heating with potassium persulfate as the oxidizingagent. The produced orthophosphate ions were then determined by thereaction 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 requireexpensive 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 doubledistilled. Risedronate sodium, 100.41% pure sample (Taha and Youssef2003), 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. forPharmaceutical and Chemical Industries, Cairo, Egypt. Tablets contain-ing risedronate sodium (Actonel tablets, batch 406767, labeled to contain5mg of risedronate sodium=tablet, Aventis Pharma Co., Cairo, Egypt)were obtained from commercial sources in a local market. Tabletscontaining etidronate disodium (Etidron tablets, batch 305741, labeledto contain 200mg of etidronate disodium=tablet, Memphis Co. for

Determination of Risedronate and Etidronate 1573

Pharmaceutical and Chemical Industries, Cairo, Egypt) were purchasedfrom commercial sources in a local market. Potassium dihydrogenorthophosphate and potassium persulfate were obtained from Winlab(Middlesex, England). Ammonium molybdate and hydrochloric acid(labeled to have purities of 98% and 32%, respectively) were obtainedfrom Merck (Darmstadt, Germany). Ammonium metavanadate (labeledto have a purity of 99%) was purchased from Aldrich Chemical Co. Ltd.(Gillingham, Dorest, England).

Molybdovanadate reagent was prepared by mixing 0.5 g ammoniummolybdate and 0.025 g ammonium metavanadate, diluting the mixturewith distilled water to about 50mL, and sonicating it in an ultrasonicbath until the solids completely dissolved. The solution initially wasintensely yellow. After 7.4mL conc. HCl was added, the yellow colorfaded away, and the mixture was diluted to 100mL with distilledwater. The solution was stable for at least 3 months when kept in therefrigerator.

All glassware was cleaned effectively with special care to avoidphosphate contamination. All glassware was thoroughly rinsed withhot 1:1 HCl and then rinsed many times with doubly distilled water.

Stock Solutions

Standard stock solutions containing 0.82mM of (I) and 1.00mM of (II)were prepared by dissolving 25.0mg of each drug in 100mL distilledwater. These solutions were stable for at least 10 days when stored inthe refrigerator and protected from light. More dilute solutions wereobtained by appropriate dilution.

Standard phosphate solutions containing 1.64mM and 2.00mM ofpotassium dihydrogen orthophosphate were prepared.

General Recommended Procedures

Procedure for Calibration Graph

Accurately measured aliquots of the stock solutions were transferred intoa series of 25-mL volumetric flasks so that the final concentrations werein the range of 0.5–10 mg=mL for (I) and 0.5–8 mg=mL for (II). Then 2 and1mL of 0.2% potassium persulfate solution for (I) and (II), respectively,were added, and the solutions were mixed well and heated in a boilingwater bath for 10min for (I) and 5min for (II). The solutions werecooled, and 0.8mL of molybdovanadate reagent was added for both

1574 M. I. Walash et al.

(I) and (II). The mixtures were shaken well and completed to volume withdistilled water. The absorbance was measured at 313 nm against anappropriate blank, prepared simultaneously. To get the standard calibra-tion graphs, the values of the absorbance were plotted against the finalconcentration in mg=mL. Alternatively, the regression equations werederived.

Procedure for Determination of the Studied Drugs in Dosage Forms

The coatings of the Actonel tablets were removed. Accurately weighedquantities of the mixed contents of 10 pulverized tablets (Actonel andEtidron), equivalent to 25.0mg of each drug, were transferred into100-mL volumetric flasks. The solutions were completed to the mark withdistilled water. The contents of the flasks were sonicated for 15min andfiltered. The nominal contents were calculated either from the previouslyplotted calibration graphs or using the corresponding regression equations.

RESULTS AND DISCUSSION

The molybdovanadate method plays a prominent role in the determina-tion of inorganic, organic phosphate (Baadenhuljsen, Seuren-Jacobs, andJansen 1977; Mas-Torres et al. 1997; Lin and Morales 1997; Ueda andWada 1970; Bartlett and Lewis 1970; Richardson 1964) and somephosphorus-containing drugs viz., alendronate sodium (Zhang et al.2000) and phosphonoformate (Forsman, Andersson, and Tornros 1986).

For determination of organic phosphate and phosphorus-containing drugs, the method is based on oxidative cleavage of thephosphorus–carbon (P–C) bond and the colorimetric determination ofthe orthophosphate ions produced after its conversion to the yellowphosphovanadomolybdate complex. It should be noted that it is notnecessary to achieve complete conversion of the analyte to measure itin pharmaceutical formulations.

The studied drugs belong to the bisphosphonate group, and theproposed method depends on their oxidation by heating with potassiumpersulfate. The generated orthophosphate ions are then determined byconversion to phosphovanadomolybdate complex, which has maximumabsorbance at 313 nm (Fig. 1).

To study the factors affecting the thermal-induced oxidation of thestudied drugs, standard phosphate solutions (1.64 and 2.00mM solu-tions) were prepared and used for the determination of the percentageconversion of the studied drugs to orthophosphate ions.

Determination of Risedronate and Etidronate 1575

Optimization of Experimental Conditions

Factors affecting the thermal-induced oxidation of the drugs as well asthe different experimental parameters affecting the color developmentand stability were carefully studied and optimized.

Effect of Potassium Persulfate Concentration

The effect of potassium persulfate concentration was studied, and theexperimental results are depicted in Fig. 2. As can be seen, maximumand constant percentage conversions (ca. 75% and 89% for (I) and (II),respectively) were achieved at volumes of 1.5 and 0.5mL of 0.2% potas-sium persulfate solution for (I) and (II), respectively, after which furtherincrease of the volume of persulfate has no effect. So, 2 and 1mL of 0.2%potassium persulfate solution were chosen as optimal for (I) and (II),respectively.

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

1576 M. I. Walash et al.

Effect of Temperature

The effect of heating temperature was studied in the range of 30–100�C.No conversion of the analytes was observed at temperatures less than50�C, whereas maximum quantitative conversion was achieved attemperatures greater than 90�C for both drugs (Fig. 3). A temperatureof 100�C was chosen as optimal for both drugs.

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

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

Determination of Risedronate and Etidronate 1577

Effect of Heating Time

The effect of heating time on the oxidation of the two drugs was studied.It was found that 7min and 3min were sufficient for maximum con-version of (I) and (II) (ca. 75% and 89%), respectively (Fig. 4). So,10min and 5min 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 0.5–1.1mLof the molybdovanadate reagent for both drugs. Larger volumes of thereagent cause a slight decrease of the absorbance (Fig. 5), noisy peaks,and increase of blank readings. So, 0.8mL was used for all experimentswithin the concentration ranges to ensure maximum absorbance andminimum blank readings.

Effect of Acidity

The effect of acidity was studied in the range of 0.4–1.6M HCl. At lowacidities, yellow-orange molybdenum–vanadium complexes were formedand led to unacceptable high-absorbing blanks; at high acidities, therate of color formation was slow, causing a decrease of the absorbanceand nonlinear response. The optimum medium for preparation of the

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

1578 M. I. Walash et al.

molybdovanadate reagent was 0.75M HCl because it gave maximumabsorbance and minimum blank readings.

Effect of Time on Stability of the Product

The absorbance of the colored complexes reached the maximum valueimmediately after the addition of the molybdovanadate reagent andremained 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 absorbancevs. the final concentrations were rectilinear over the concentration rangescited in Table 1. Linear regression analysis of the data gave the followingequations:

Risedronate: A ¼ 0:0018þ 0:1127C ðr ¼ 0:9999ÞEtidronate: A ¼ 0:0273þ 0:1482C ðr ¼ 0:9999Þ

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

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

Determination of Risedronate and Etidronate 1579

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

Limit of Quantitation and Limit of Detection

The limit of quantitation (LOQ) was determined by establishing theweakest concentration that can be measured according to the ICHQ2(R1) recommendation (ICH 2005), below which the calibrationgraph 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 deter-mined by evaluating the weakest concentration of the analytes thatcan be readily detected and was found to be 0.094 and 0.134 mg=mL(3.081� 10�7 and 5.36� 10�7 M=L) for (I) and (II), respectively.

The LOQ and LOD were calculated according to the followingequations (ICH 2005):

LOQ ¼ 10Sa=b

LOD ¼ 3:3Sa=b

Table 1. Analytical data for the proposed method

Parametera Risedronate Etidronate

Concentration range (mg=mL) 0.5–10 0.5–8Limit of detection (LOD) (mg=mL) 0.094 0.134Limit of quantification (LOQ) (mg=mL) 0.286 0.405Correlation coefficient 0.9999 0.9999Slope 0.1127 0.1482Intercept 0.0018 0.0273Sy=x 4.09� 10�3 6.55� 10�3

Sa 3.22� 10�3 6.00� 10�3

Sb 4.59� 10�4 9.67� 10�4

RSD (%) 0.96 1.02Error (%) 0.37 0.46e (l=mol.=cm.) 3.482� 104 3.948� 104

aSy=x: standard deviation of the residuals; Sb: standard deviation ofthe slope; Error¼% RSD=

pn; Sa, standard deviation of the inter-

cept; and E, molar absorbtivity.

1580 M. I. Walash et al.

Table

2.Accuracy

andprecisiondata

forthestudieddrugsusingtheproposedmethod

Parameter

Intradayprecision

Interdayprecision

Concentration

added

(mg=mL)

Concentration

found

(mg=mL)

Recovery

(%)

Concentration

added

(mg=mL)

Concentration

found

(mg=mL)

Recovery

(%)

Risedronate

4.0

3.974

99.35

4.0

4.043

101.08

6.0

5.929

98.81

6.0

5.963

99.38

8.0

8.030

100.40

8.0

7.930

99.14

x�SD

99.52�0.81

99.87�1.06

RSD

(%)

0.81

1.06

Error(%

)0.47

0.61

Etidronate

1.0

0.995

99.50

1.0

0.9815

98.15

4.0

4.054

101.35

4.0

4.017

100.43

6.0

5.968

99.47

6.0

5.968

99.47

x�SD

100.11�1.08

99.35�1.15

%RSD

1.08

1.16

%Er

0.62

0.67

Note.Each

resultistheaverageofthreeseparate

assays.

1581

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

Accuracy and Precision

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

Table 3. Application of the proposed and comparison methods to the assay of thestudied drugs in active pharmaceutical ingredient and reference forms

Parameter

Proposed method Comparison method

Conc.added

(mg=mL)

Conc.found

(mg=mL)Recovery

(%)

Conc.added

(mg=mL)Recovery

(%)

Risedronate0.5 0.490 98.001.0 0.995 99.50 4.0 99.902.0 1.981 99.05 8.0 101.384.0 4.004 100.10 10.0 99.966.0 6.060 101.008.0 8.021 100.2610.0 9.956 99.56

x� SD 99.64� 0.96 100.41� 0.84t 1.201 (2.306)�

F 1.306 (19.33)�

Etidronate2.0 1.996 99.804.0 4.078 101.95 4.0 98.985.0 4.978 99.56 8.0 101.666.0 6.024 100.40 10.0 99.188.0 7.958 99.48

x� SD 100.24� 1.02 99.94� 1.49t 0.401 (2.447)�

F 2.134 (19.25)�

Note. Each result is the average of three separate assays.�Values between brackets are the tabulated t and F values at p¼ 0.05.

1582 M. I. Walash et al.

APPLICATION OF THE PROPOSED METHOD

The proposed method was applied to the determination of thestudied drugs in active pharmaceutical ingredients, reference forms,and tablets. The percentage recoveries of the studied drugscompared with those obtained by the comparison method (Tahaand Youssef 2003) are given in Tables 3 and 4. The comparisonmethod involved the spectrophotometric determination of the twodrugs by oxidation with ceric(IV) sulfate in 0.5M sulfuric acidand subsequent measurement of the excess unreacted ceric(IV)sulfate at 320 nm.

Statistical analysis (Miller and Miller 2005) of the results obtained bythe proposed and comparison methods using Student’s t-test and var-iance ratio F-test revealed no significant differences between the accuracyand precision of the two methods.

Table 4. Application of the proposed and comparison methods to the assay of thestudied drugs in dosage forms

Parameter

Proposed method Comparison method

Conc.added

(mg=mL)

Conc.found

(mg=mL)Recovery

(%)

Conc.added

(mg=mL)Recovery

(%)

Actonel tablets (5mg risedronate sodium=tablet)2.0 1.972 98.60 4.0 100.384.0 3.960 99.00 8.0 99.956.0 6.015 100.25 10.0 102.27

x� SD 99.28� 0.86 100.87� 1.23t 1829 (2.776)�

F 2.046 (19.00)�

Etidron tablets (200mg etidronate disodium=tablet)1.0 1.018 101.80 4.0 100.604.0 4.011 100.28 8.0 99.096.0 5.956 99.16 10.0 99.04

x� SD 100.08� 0.84 99.58� 0.89t 0.969 (2.776)�

F 1.123 (19.00)�

Note. Each result is the average of three separate assays.�Values between brackets are the tabulated t and F values at p¼ 0.05.

Determination of Risedronate and Etidronate 1583

MECHANISM OF THE REACTION

First Step

The first step involved oxidation of the studied drugs by heating withpotassium persulfate as the oxidizing agent, yielding orthophosphate ionsas shown in Scheme 2.

Second Step

In the second step of the reaction, the generated orthophosphate ionsreacted with ammonium molybdate and ammonium metavanadate toyield phosphovanadomolybdate complex, which has a yellow colormeasured at 313 nm.

CONCLUSION

A simple and sensitive spectrophotometric method was developed fordetermination of etidronate and risedronate in either active pharmaceuticalingredients or commercial tablets. Compared to previously reported chroma-tographic methods for the determination of the studied drugs, our method issimple and inexpensive. Additionally, it does not require the elaborate proce-dures associated with chromatographic methods. In comparison to otherreported spectrophotometric methods currently used for determination ofthe studied drugs, our method is superior in terms of sensitivity and speed.

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

1584 M. I. Walash et al.

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