Você está na página 1de 6

Lamie N T et al.

/ Pharmacie Globale (IJCP) 2011, 7 (06)

Available online at www.pharmacie-globale.info

ISSN 0976-8157

Research Article

PHARMACIE GLOBALE INTERNATIONAL JOURNAL OF COMPREHENSIVE PHARMACY

STABILITY INDICATING METHODS FOR THE DETERMINATION OF SILDENAFIL CITRATE IN THE PRESENCE OF ITS DEGRADATION PRODUCT
Amr M Badawy, Abd El-Aziz B Abd El-Aleem and Nesrine T Lamie*
Department of Analytical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, ET 11562, Cairo, Egypt. Received: 24 April 2011; Revised: 27 June 2011; Accepted: 30 June 2011; Available online: 5 July 2011

ABSTRACT

Three stability-indicating methods were developed for the determination of Sildenafil Citrate (SLDC) in the presence of its degradation product. The First method depends on the quantitative densitometric evaluation of thin layer chromatogram of sildenafil citrate in the presence of its degradation product without any interference. Chloroform : methanol : ammonia (9: 1: 0.01 by volume) was used as a mobile phase and the chromatogram was scanned at 292 nm. This method determines SLDC in the concentration range 1.2-3.6 g/spot with mean percentage recovery 99.800.928%. The second method is based on the high performance liquid chromatographic separation on Phenomenex10 ODS (150 mm 3.9 mm I.D), particle size (10m), with a mobile phase consisting of acetonitrile: methanol: 0.05 M potassium dihydrogen phosphate (pH: 5.8) (50: 20: 30 v/v/v followed by UV detection at 290nm. Calibration graph is linear in the concentration range 8-40g/ml with mean percentage recovery of 100.231.188 %. The third method depends on application of bivariate calibration algorithm to the spectrophotometric determination of SLDC in the concentration range 10-60 g/ml with mean percentage recovery100.051.181%. The suggested procedures were checked 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 reference method. Keywords: Sildenafil citrate, TLC densitometry, HPLC, Bivariate spectrophotometric method.

INTRODUCTION

Sildenafil citrate 1-[[3-(6,7-Dihydro -1-methyl- 7-oxo-3propyl -1H-pyrazolo [4,3-d] pyrimidin-5-yl) -4ethoxyphenyl]sulphonyl]-4-methyl piperazine citrate (Figure 1) is a compound of the pyrazolo-pyrimidinylmethyl piperazine class, and is used to treat male erectile dysfunction. It is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 inhibitor.1 Sildenafil citrate could be determined by several analytical techniques, Densitometry1, spectrophotometry2, colorimetry3-6, HPLC7-18, GC-MS19, MEKC20, 21 and capillary electrophoresis22,23. Figure 1. Structural formula of Sildenafil citrate

methods for the determination of SLDC in the presence of its oxidative degradation product with satisfactory statistical validation measures.

MATERIALS AND METHODS

Chemicals and Reagents All chemicals and reagents were of analytical grade unless otherwise stated and distilled deionised water was used. Chloroform (Merck), Hydrogen peroxide 30%, Ammonium hydroxide (Adwic), acetonitrile (Merck, HPLC grade) methanol (Merck, HPLC grade), 0.05 M potassium dihydrogen phosphate (Sigma, pH: 5.8).

Pure samples: Sildenafil citrate was kindly supplied by Amoun company, It was analyzed and found to be 100.170.862 by applying RP- HPLC method.16 Market samples: Vigoran tablets, produced by Amoun Parmaceuticals, Batch number: 6207. Each tablet was labelled to contain sildenafil citrate equivalent to 50 mg sildenafil citrate.

The aim of this work was to develop stability indicating


*Corresponding Author: Dr Nesrine Talaat Lamie Department of Analytical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, ET 11562, Cairo, Egypt. Contact no: +20-122711259, Email: nesrinelamie@hotmail.com

Preparation of stock solutions Sildenafil citrate stock standard solution (0.1mg/ml) was prepared by weighing accurately 10mg of sildenafil citrate into 100 ml volumetric flask, and then diluted to the volume with methanol. Degradation product stock solution (0.1mg/ml) in methanol. Fenofibrate (internal standard) 0.1 mg/ml in methanol. Pharmacie Globale (IJCP), Vol. 02, Issue 07

Lamie N T et al. / Pharmacie Globale (IJCP) 2011, 7 (06)

Instrumentation UV/VIS spectrophotometer (SHIMADZU 1601/PC). Shimadzu-Dual wavelength flying spot CS- 9000 densitometer, Hamilton syringe 25l, Camag TLC scanner 35/N/30319 with WINCATS software. UV lamp-short wavelength at 254nm (Desaga, Germany). Thin-layer chromatographic plates precoated with silica gel G.F254 20x20cm, 0.25mm thickness (E Merck, Darmstadt, Germany). HPLC system consists of Agilent pump with different flow rates (model 1100 series, Agilent, USA), equipped with a variable wavelength detector and a 20 l volume injection loop. Phenomene x 10 ODS (150mm 3.9mm I.D); particle size (10m) was used as stationary phase. The samples were injected with 50l Hamilton analytical syringe. 6-WPA pH combined electrode model C D 740 was used for pH measurements. Preparation of degradation product An accurately weighed amount of pure authentic sildenafil citrate (200mg) was dissolved in 30 ml methanol in a 250ml flask. Two ml of 30% H2O2 solution were added and then refluxed for 6 hours. The methanolic solution was heated on a boiling water bath to get rid of excess H2O2 then evaporated under vaccum and dried in a dessicator. The degradate identity was checked and confirmed by mass spectroscopy and IR. Complete degradation was confirmed by spotting onto TLC plate, and using Chloroform : methanol : ammonia (9: 1: 0.01v/v/v) as a developing system in a presaturated chromatographic tank for 30 minutes. By using ascending chromatography for 20 minutes, the spots were dried and visualized under UV light at 254 nm. Laboratory-prepared mixtures TLC-Densitometric method- Aliquots of 2.4 to 1.2 mg were accurately transferred from SLDC stock standard solution (0.1 mg/ml) into 10ml volumetric flasks. To the previous solutions aliquots of 1.2 to 2.4 mg of SLDC degradate stock standard solution (0.1mg/ml) were added, the total volume was completed to the mark with methanol. For HPLC method- Aliquots equivalent to 32-8g/ml were accurately transferred from SLDC stock standard solution (0.1 mg/ml) into 10ml volumetric flasks. To the previous solutions aliquots equivalent to 8-32g/ml of SLDC degradate stock standard solution (0.1mg/ml) were added, then 1ml of internal standard fenofibrate was added and the volume was completed to the mark with methanol. Bivariate method- Aliquots of 48.0 to 12.0 ml were accurately transferred from SLDC stock standard solution (0.1 mg/ml) into 10ml volumetric flasks. To the previous solutions aliquots of 12.0 to 48.0 ml of SLDC degradate stock standard solution (0.1 mg/ml) were added and the volume was completed to the mark with methanol. TLC-densitometric method Linearity: Aliquots of SLDC stock standard solution (0.1mg/ml) 12, 16, 20.36l equivalent to (1.2-3.6g) were applied onto a TLC plate using a 100l syringe. Spots were spaced 1.5cm apart from each other and from the bottom edge of the plate. The plates were developed in a chromatographic tank previously saturated with the mobile phase, chloroform : methanol : ammonia (9: 1: 0.01 v/v/v) by ascending technique through a distance of 8 cm at room temperature. The plate was removed, air dried and the spots were visualized under UV lamp at 254nm.

The chromatogram was scanned at 292nm. The calibration curve representing the relationship between the recorded area under the peak and the corresponding concentration was plotted and the regression equation was computed. Assay of laboratory-prepared mixtures: The chromatographic conditions were applied for each laboratory-prepared mixture and the concentration of SLDC in these mixtures was calculated by substituting in the regression equation. HPLC method Linearity: Aliquots of SLDC stock standard solution (0.1mg/ml) equivalent to (8-40 g/ml) were accurately transferred into a series of 10ml volumetric flasks; the volume was completed to the mark with methanol. 20 l of each solution was injected on Phenomenex10 ODS (150mm 3.9mm I.D), particle size (10m), using a mobile phase consisting of acetonitrile: methanol: 0.05 M potassium dihydrogen phosphate (pH: 5.8) (50: 20: 30 v/v/v), at flow rate 0.7 ml/min and detection at 290nm.The calibration curve was constructed relating the peak area ratios of SLDC to that of fenofibrate versus the corresponding concentration of SLDC and The regression equation was computed.
A= 0.0302 C -0.0212 r = 0.9991

Where A is the concentration of SLDC in g/ml and r is the correlation coefficient. Assay of laboratory prepared mixtures: The chromatographic conditions were applied for each laboratory-prepared mixture and the concentration of SLDC in these mixtures was calculated by substituting in the regression equation. Bivariate method Aliquots of SLDC stock standard solution (0.1 mg/ml) equivalent to (600-100g) and aliquots of SLDC degradate stock standard solution (0.1 mg/ml) equivalent to (100600g) were accurately transferred separately into 10-ml volumetric flasks and the volume was completed to the mark with methanol. The absorbances were recorded in the range of 200-400 nm for each set. The calibration curves were constructed at different wavelengths 250, 270, 290, 300, 310, 320 nm. The regression equations were computed at each wavelength. From both sets of regression equations, the K values were calculated and the maximum value of K was chosen corresponding to two wavelengths at which the laboratory prepared mixtures were measured. Application to Vigoran tablets Five tablets were accurately weighed and finely powdered. A portion equivalent to 10mg of SLDC was weighed, sonicated in 30ml methanol and filtered into a 100ml volumetric flask. The residue was washed three times each with 10ml methanol and completed to the mark with the same solvent. Aliquots (according to linearity) were transferred to 10ml volumetric flasks and diluted with methanol for TLC-densitometric and HPLC methods. The general procedure was followed and the concentration of SLDC was calculated from its corresponding regression equation. Bivariate method: 2ml of the filtrate solution were accurately transferred into a 10ml volumetric flask and 4ml of degradation stock solution were added. The volume was completed to the mark with methanol. The Pharmacie Globale (IJCP), Vol. 02, Issue 07

Lamie N T et al. / Pharmacie Globale (IJCP) 2011, 7 (06)

absorbance was recorded at 250 and 290 nm .The concentration of SLDC in Vigoran tablets was calculated by applying Kaisers equation.

RESULTS AND DISCUSSION

method was applied for the determination of pure drug and satisfactory results were obtained (Table 1). Figure 2. Suggested pathway for the degradation of sildenafil citrate.

Analytical methods for the determination of a compound in presence of its degradate without previous chemical separation are always of interest. Sildenafil citrate was subjected to different drastic conditions of acidity (1 N HCl) and alkalinity (1 N NaOH). However, it was found to withstand degradation under these conditions. The degradation of sildenafil citrate was achieved by refluxing SLDC with 30% H2O2 for 6 hrs. The degradate was separated by TLC, using chloroform: methanol: ammonia (9:1:0.01v/v/v) as a mobile phase and visualized under UV lamp at 254nm. The Rf values of SLDC and its degradate were 0.62 and 0.11 respectively. The suggested structure of the degradation product was confirmed by IR analysis where, the spectrum of SLDC-degradate showed broadening and increase in the peak intensity at 3450cm-1. This may be attributed to the association of the formed sulphonic acid group (SO3H).Also the expected molecular weight of degradate was found to be 537 which was confirmed by mass spectrometery. The suggested reaction of degradation is shown in Figure 2. TLC-Densitometric method In this work, TLC-densitometric method was used for the determination of SLDC in presence of its degradate, depending on the difference in Rf values. Different developing systems were tried, but complete separation of SLDC and its degradate was achieved using chloroform: methanol: ammonia (9:1:0.01v/v/v) as a mobile phase. Densitometric scanning was performed at max 292nm with excellent results (Figure 3).

Figure 3. Scanning profile of the TLC chromatogram of sildenafil citrate (1.2-3.6 g/spot) at 292 nm using Chloroform : methanol : ammonia (9: 1: 0.01 by volume) as a mobile phase.

A linear correlation was obtained between the peak area of the spots against the corresponding concentration, the parameters of the regression equation and the concentration range are shown in (Table 1). The proposed Table 1. Regression equation parameters and results of determination of pure sample of sildenafil citrate by the proposed methods.
Parameter TLC-Densitometric method HPLC method Bivariate method Accuracy (mean S.D.) 99.800.928 100.231.188 100.051.181 Specificity 99.250.976 99.861.623 101.000.988 Precision Repeatability* 100.690.783 100.711.197 100.690.711 Intermediate precision** 99.160.962 98.871.234 98.870.993 Linearity Slope 0.4131 0.0302 0.0196 Standard error of the Slope 0.0052 0.000407 0.000245 Intercept 0.886 -0.0212 0.0047 Standard error of the Intercept 0.0132 0.010972 0.005904 Correlation coefficient (r) 0.9992 0.9991 0.9994 Range 1.2-3.6 g/spot 8-40g/ml 10-60 *The intraday and **the inter-day mean values standard deviations of samples of concentration of 2, 2.8, 3.6 g/spot of sildenafil citrate for TLCdensitometric method, 10, 15, 20 g/ml for HPLC method and 20, 30, 40 g/ml for Bivariate method.

The laboratory-prepared mixtures were analysed by TLCdensitometric technique. The method is valid for determining the drug in laboratory-prepared mixtures containing up to 70% degradation product as shown in (Table 2). HPLC method Good chromatographic separation of SLDC and its degradate could be achieved by using Phenomenex10 ODS (150 mm 3.9 mm I.D), particle size (10m), with a mobile phase consisting of acetonitrile: methanol: 0.05 M potassium dihydrogen phosphate (pH: 5.8) (50: 20: 30 v/v, followed by UV detection at 290 nm, (Figure 4).

Table 2. Determination of sildenafil citrate in laboratory prepared mixtures by the proposed methods.
Mixture No. 1 2 3 4 5 6 Mean S.D. R.S.D.% TLC-Densitometric method Found % 98.05 100.48 98.62 99.94 99.17 99.25 0.976 0.983 HPLC method Found % 102.05 99.53 98.34 98.96 98.59 101.62 99.86 1.623 1.625 Bivariate method Found % 101.40 101.71 99.31 101.17 102.00 100.42 101.00 0.988 0.978

Pharmacie Globale (IJCP), Vol. 02, Issue 07

Lamie N T et al. / Pharmacie Globale (IJCP) 2011, 7 (06)

Figure 4. HPLC chromatogram of sildenafil citrate (20 g/ml; Rt: 3.21min), its degradation product (20g/ml; Rt :4.01min) and its internal standard (fenofibrate; Rt: 6.74 min).

Bivariate method According to Kaisers method, the slope values of the linear regression equations for both components at the selected wavelengths were used to calculate K values (sensitivity matrices) to find out the optimum pair of wavelengths at which the binary mixtures are recorded.24 For the binary mixture of sildenafil citrate and its degradation product 250 and 290 nm are found to give the maximum value of K as shown in (Table 4). Table 4. Application of Kaisers method in the selection of wavelength pair for the mixture of sildenafil citrate and its degradate: the absolute values of determinants of sensitivity matrices(K10-5).
/ 250 270 290 300 310 320 250 0 270 9.10 0 290 14.85 -0.96 0 300 8.51 -2.26 -4.58 0 310 1.01 -4.94 -8.17 -4.37 0 320 -1.75 -4.55 -7.24 -4.69 -1.45 0

*(Rt :6.74min) using the specified chromatographic conditions. Mobile phase: acetonitrile : methanol: 0.05 M potassium dihydrogen phosphate (pH: 5.8) (50: 20: 30 v/v/v). Detection: 290nm.

Several trials have been undertaken to reach the optimum stationary/mobile phases matching. The suggested chromatographic system allows complete base line separation at reasonable time. In (Table 3) statistical analysis of the parameters required for system suitability test of HPLC method were shown, indicating good resolution of the two components. Table 3. Parameters required for system suitability test of HPLC method
Parameter Resolution (R) T (tailing factor) (relative retention time) K (column capacity) N (column efficiency) HETP Obtained value 1.14 Sildenafil citrate 1.06 Deg. product 1.66 1.615 Sildenafil citrate 0.681 Deg. product 1.099 Sildenafil citrate 257.6 Deg. product 714.6 Sildenafil citrate 0.058 Deg. product 0.021 Reference value R > 0.8 T = 1 for a typical symmetric peak >1 Till 10 acceptable Increases with efficiency of the separation The smaller the value, the higher the column efficiency

The linear regression calibration formulae used for the bivariate algorithm are presented in (Table 5).The concentration is calculated by compensating in the following equation:

Where,

A linear correlation was obtained between the peak area Calibration Equation ratio against the corresponding concentrations, the Component =250nm =290nm parameters of the regression equation and the Sildenafil A =0.0232x+0.0015 A=0.0196x+0.0047 concentration range are shown in (Table 1). The proposed citrate (r =0.9992) (r =0.9994) method was applied for the determination of pure drug A =0.0277x +0.0094 A =0.017x+0.0032 and satisfactory results were obtained (Table 1). The Degradate (r =0.9997) (r =0.9997) laboratory prepared mixtures were analyzed by HPLC The proposed methods were also successfully applied for method. The method is valid for determining the drug in the analysis of the drug in pharmaceutical dosage forms laboratory-prepared mixtures containing up to 70% and the results are shown in (Table 6). degradation product as shown in (Table 2). Table 6. Quantitative determination of sildenafil citrate in Vigoran tablets by the proposed bivariate method and results of application of standard addition technique.
TLC-Densitometric method Vigoran tablets Claimed taken* (g/ml) Pure added (g/ml) 1.20 1.60 2.00 2.40 Pure found (g/ml) 1.21 1.63 2.02 2.41 Recovery (%) 100.83 101.88 101.00 100.42 101.03 0.615 0.609 Claimed taken* (g/ml) HPLC method Pure added (g/ml) 8.00 10.00 15.00 20.00 25.00 Pure found (g/ml) 7.95 10.17 15.16 19.84 25.04 Recovery (%) 99.38 101.70 101.07 99.20 100.16 100.30 1.076 1.073 Claimed taken* (g/ml) Bivariate method Pure added (g/ml) 10.00 15.00 20.00 30.00 40.00 Pure found (g/ml) 10.08 14.87 20.25 30.41 39.63 Recovery (%) 100.80 99.13 101.25 101.37 99.08 100.33 1.135 1.131

CA= Concentration of component A (Drug). CB= Concentration of component B (Degradate). mA1= slope for component A (Drug) at 1. mA2= slope for component A (Drug) at 2. mB1= slope for component B (Degradate) at 1. mB2= slope for component B (Degradate) at 2. AAB1= Absorbance of binary mixture at 1. AAB2= Absorbance of binary mixture at 2. eAB1= Sum of intercepts for both components A & B at 1. eAB2: Sum of intercepts for both components A &B at 2. Table 5. Linear regression calibration formulae used for the bivariate algorithm for sildenafil citrate.

Batch No. 6207

1.19

10.12

20.14

Mean S.D. R.S.D% *Average of three different determinations.

Pharmacie Globale (IJCP), Vol. 02, Issue 07

Lamie N T et al. / Pharmacie Globale (IJCP) 2011, 7 (06)

The validity of the proposed methods was assessed by method.16 Calculated t and F values are less than the applying the standard addition technique. The results theoretical ones, indicating with 95% confidence limit that obtained were reproducible with low relative standard there is no significant difference between the proposed deviation as shown in (Table 6). Table 7 showed the and the reference methods with respect to accuracy and statistical comparison of analytical results for pure precision. samples by the proposed method and the reference Table 7. Statistical analysis of the results obtained by the proposed methods and the reference method for the analysis of sildenafil citrate in pure powder form.
Item TLC-Densitometric method HPLC method Mean 99.80 100.23 S.D. 0.928 1.188 Variance 0.861 1.411 n 7 7 Students t test 0.664 (2.228)** 0.010(2.228)** F value 1.159 (6.160)** 1.899(6.160)** *RP- HPLC method **The figures in parenthesis are the corresponding tabulated t and F values at P=0.05. Bivariate method 101.00 0.988 0.976 6 1.419 (2.262)** 1.314 (6.260)** Reference method*(16) 100.17 0.862 0.743 5

CONCLUSION

The suggested methods can be used as stability-indicating methods for determination of bulk powder or pharmaceutical formulations of SLDC without interference from its degradation product or from excipients. In

addition to their stability indicating ability, the proposed methods are more simple, fast, saving time, require minimum chemicals, sensitive, accurate, precise, and can be used for quality control laboratories.

REFERENCES

1. Badwan A A, Nabulsi L, Al-Oman M M, Darghmeh N and Ashour M; In "Analytical profiles of drug substances and excipients", vol. 27, edited by Florey, K., Academic Press. 2001; 340-376. 2. Shaimaa M Badr; Thesis of M Pharm Sci, Faculty of Pharmacy, Cairo Univ. 2004. 3. Amin A S, Moustafa M E, El-Dosoky R; Colorimetric determination of sildenafi l citrate (Viagra) through ion-associate complex formation. J AOAC Int. 2009 JanFeb; 92(1):125-30. 4. Jiang H, Zhan H L, Wu X F; Fenxi Kexue Xuebao. Jun 2004; 20(3):287-289. 5. Qin Z H, Tan R; Fenxi Kexue Xuebao, Apr 2004; 20(2):175-177. 6. Reddy M N, Murthy T K, Rao Y S, Sushma K, Sankar D G; Extractive spectrophotometric determination of sildenafil citrate in pharmaceutical dosage forms. Indian Drugs. Feb. 2002; 39(2):106-109. 7. Cooper J D H, Muirhead D C, Taylor J E, Baker P R; Development of an assay for the simultaneous determination of sildenafil (Viagra) and its metabolite (UK-103,320) using automated sequential trace enrichment of dialysates and high-performance liquid chromatography. J Chromatogr B Biomed Sci Appl. 1997; 701:87-95. 8. Segall A I, Vitale M F, Perz V L, Palacios M L and Pizzorno M T; Reversed-phase HPLC determination of sildenafil citrate in the presence of its oxidativeinduced degradation products. J Liq Chromatogr Relat Technol. 2000; 23(9):1377-1386. 9. Abd-ELbary A, Foda N H, El-Gazayerly O N; Stabilityindicating high-performance liquid chromatographic assay for the determination of sildenafil citrate in bulk and in formulations. Chromatographia. May 2004; 59(9-10):561-566. 10. Jung B H, Moon D G, Chung B C; HPLC determination of sildenafil and its demethylated metabolite, UK-103,320, in cat plasma with solid-phase extraction for the pharmacokinetic study. Can J Anal Sci Spectrosc. 2001; 46(2):40-45. 11. Guermouche M H, Bensalah K; Solid phase extraction and liquid chromatographic determination of sildenafil

and N-demethylsildenafil in rat serum with basic mobile phase. J Pharm Biomed Anal. Mar 2006; 40(4):952-957. 12. Liu Y M, Yang H C, Miao J R, Yaowu Fenxi Zazhi; 2000; 20:61. Through "Analytical Abstracts". 2000; 62(6):6G204. 13. Daraghmeh N, Al-Omari M, Badwan A A and Jaber A M; Determination of sildenafil citrate and related substances in the commercial products and tablet dosage form using HPLC. J Pharm Biomed Anal. 2001; 25(3-4):483. 14. Aboul-Enein H Y, Hefnawy M M; Rapid Determination of Sildenafil Citrate in Pharmaceutical Preparations Using Monolithic Silica HPLC Column. J Liq Chromatogr Relat Technol. Oct 2003: 26(17):2897-2908. 15. Altiokka G, Atkosar Z, Sener E and Tuncel M; FIA of sildenafil citrate using UV-detection. J Pharm Biomed Anal. 2001; 25(2):339-342. 16. Dinesh N D, Vishukumar B K, Nagaraja P, Made Gowda N M and Rangappa K S; Stability indicating RP-LC determination of sildenafil citrate (Viagra) in pure form and in pharmaceutical samples. J Pharm Biomed Anal. 2002; 29(4):743-748. 17. Jeong C K, Lee H Y, Jang M S, Kim W B and Lee H S; Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching. J Chromatogr B. 2001; 752(1):141-147. 18. Sheu M T, Wu A B, Yeh G C, Hsia A and Ho H; Development of a liquid chromatographic method for bioanalytical applications with sildenafil. J Chromatogr B. 2003; 791:255-262. 19. Sashio K, Scott K S, Morimoto S and Nakahara Y; Hair analysis for pharmaceutical drugs. II. Effective extraction and determination of sildenafil (Viagra) and its N-desmethyl metabolite in rat and human hair by GC-MS. Biol Pharm Bull. 2001; 24(12):1384-1388. 20. Berzas Nevado J J, Rodriguez Flores J, Castaneda Penalvo G and Rodriguez Farinas N; Micellar electrokinetic capillary chromatography for the determination of Viagra and its metabolite (UK103,320) in human serum. Electrophoresis. 2001; Pharmacie Globale (IJCP), Vol. 02, Issue 07

Lamie N T et al. / Pharmacie Globale (IJCP) 2011, 7 (06)

22(10):2004-2009. 21. Berzas Nevado J J, Rodrguez Flores J, Castaeda Pealvo G, Mora Diez N; Development of a Micellar electrokinetic capillary chromatography method for the determination of three drugs employed in the erectile dysfunction therapy. J Chromatogr B Analyt Technol Biomed Life Sci. Nov 25, 2004; 811(2):231-236. 22. Li Run-Kai, Bo Tao, Liu Hu-wei, Li Ke-an; Determination of Sildenafil in Medicines for Erectile Dysfunction by Capillary Electrophoresis. Chinese Journal of

Chromatography. 2002; 20(4):335-337. 23. Zhang Xinrong, Chan Yifeng, Wu Xiang-feng, Liu Lili, Yang, Genyin Dier Junyi Daxue Xuebao. 2001; 22(5):427-428. 24. Lopez-de-Alba P, Wrobel K, artinez L, Wrobel K, Marrieta M and Heranez J; Application of The Bivariate Spectrophotometric Method for The Determination of Metronidazole, Furazolidone and DiIodohydroxyquinoline in Pharmaceutical Formulations. J Pharm Biomed Anal. 1997; 16:349-355.

Pharmacie Globale (IJCP), Vol. 02, Issue 07