- Email: [email protected]
The polarographic behaviour of ketoprofen and assay of its capsules using spectrophotometric and voltammetric methods
Tahta, Vol. 41, No. 5, pp. 639-645,1994
Copyright 0 1994Elscvicr Science Ltd Printed in GreatBritain. All rightsresewed 0039-9140/94$7.00+ 0.00
Pergamon
THE POLAROGRAPHIC BEHAVIOUR OF KETOPROFEN AND ASSAY OF ITS CAPSULES USING SPECTROPHOTOMETRIC AND VOLTAMMETRIC METHODS KAMLA M. EMARA,* Azw
M. M. ALI
and NAGWA ABG-EL MAALI
Department of Analytical Pharmaceutical Chemistry, Faculty of Pharmacy and Department of Chemistry, Faculty of Science, University of Assiut, As&t, Egypt (Received 15 December 1992. Revised 21 September 1993. Accepted 21 September 1993) Summary-The quantitative determination of ketoprofen using spectrophotometric and voltammetric methods are described. The spectrophotometric procedure depends upon the reaction of ketoprofen with N-bromosuccinimide (NBS). The residual reagent is then determined by formation of violet colour with 2,2-diphenyl-1-picryl hydrazine (DPPH,). The consumed NBS would correspond to ketoprofen. Beer’s law is valid over the concentration range 5-80 rg/ml of the drug. Direct current (DC) polarography allows to study the reduction behaviour of ketoprofen at the dropping mercury electrode @ME) using different supporting electrolytes at different pH values. Direct current stripping voltammetry (DCSV) was used for the quantitative measurements of the drug. The calibration graph of peak current vs concentration was linear from 0.254 x lo-* to 0.254 &ml. In model solutions as little as 5.08 x low4 ng/ml ketoprofen can be detected by DCSV. Both methods were applied successfully for the determination of ketoprofen either in pure or dosage forms.
Ketoprofen 2-(3-benzoylphenyl) propionic acid (I), is an anti-inflammatory and analgesic drug. It is used in the treatment of rheumatoid arthritis and osteoarthritis.‘** cH3 I
Scheme 1.
The compendia1 method for the determination of authentic drug involves titration with standard sodium hydroxide in aqueous ethanolic medium, while ketoprofen in capsules has been determined by U.V. spectrophotometry at 258 nm after extraction with methanol (75%).3 Existing analytical procedures for the assay of ketoprofen include potentiometric titration,4 ultraviolet spectrophotometry,“5 visible spectrophotometry,“’ polarography,%’ gas or liquid chromatography4*‘0 and high-performance liquid chromatography. ‘I Reported methods for its *Author for correspondence.
determination in biological samples include spectrophotometry,‘* calorimetry or polarography,i3 thin-layer or paper chromatography” and high-performance liquid chromatography.“6 Although the nitrophenylhydrazine method9 and the coloured ion pairs (with basic dyes) methodsa yielded comparable results, the spectrophotometric method proposed was found to be preferable for the analysis of capsules because it was not as tedious as the nitrophenylhydrazine method and does not require extraction prior to spectrophotometric measurements. Moreover, the method is more convenient than the B.P. (1988) method for the assay of ketoprofen powder as only a small amount of the drug is necessary for each analysis. Stripping voltammetry is an important technique for trace determination of many inorganic and organic substances.lg The adsorptive strip ping technique has been used successfully for determination of subnanogram level of several drugs.20v2’ The present work deals with the quantitative determination of ketoprofen using direct current stripping voltammetry as well as spectrophotometric methods. In addition the polarographic behaviour of the drug was investigated. Both methods are simple, rapid, sensitive, reproducible and easy to apply to routine usage. 639
640
KAMLA
M. EMARAet al.
EXPERIMENTAL
Materials and reagents
Ketoprofen: supplied by Alexandria Pharm. Co. Alexandria, Egypt. NBS (SIGMA, U.S.A.) 1 x 10-3M (0.178 mg/ml) in distilled water. 2,2 - Diphenyl - 1 - picrylhydrazine (DPPH,) (SIGMA, U.S.A.), 1 x 10m3M (0.395 mg/ml) was freshly prepared in ethanol. 2,2 - Diphenyl - 1 - picrylhydrazyl (SIGMA, U.S.A.), 1 x 10p3M (0.394 mg/ml) was freshly prepared in ethanol. Supporting electrolytes: 0.1 M 0.1 M l O.lM l O.lM ment lytes.
l l
l
0.1N
perchloric acid. phosphoric acid. nitric acid. sodium hydroxide; used for adjustof the pH of the supporting electroacetic acid-sodium
acetate buffer.
All chemicals and solvents were of analytical grade. Instrumentation
Uvidec-320 spectrophotometer (JASCO, Tokyo, Japan). l Polarograph PRG 5 (Tacussel, France) equipped with three electrode potentiostatic system. Dropping mercury electrode @ME) has the capillary characteristics m = 2.10 mg/sec, t = 4.11 set (open circuit) and m 2/3t‘I6= 2.07 mg2/3/sec”2. l For voltammetric studies: an EG & G Princeton Applied Research Corp. (PAR) Model 264A was used, coupled with a PAR 303A Static Mercury Drop Electrode (SMDE) (drop size: medium, area of the drop: 0.014 cm*). The polarographic cell (PAR Model KOO60) was fitted with an Ag/AgCl saturated KCl, reference electrode and a platinum wire counter electrode. A PAR 305 stirrer was connected to the 303A SMDE and a PAR Model REOOS9 X-Y recorder was used for the collection of the experimental data. l
Method I. Spectrophotometric method Preparation of sample solutions Ketoprofen powder. Weigh accurately 25 mg of
ketoprofen and dissolve in 100 ml ethanol. Use 2-ml aliquot of this solution for the procedure. Capsules. Shake a quantity of the mixed con-
tents of 20 capsules containing 25 mg of ketoprofen for 10 min with 50 ml ethanol. Filter and wash the residue with sufficient ethanol to give a volume of 100 ml. Use 2 ml aliquot of this solution for the procedure. Procedure.
Pipette 2.0 ml of the sample solution into a 10 ml calibrated flask. Add 0.5 ml of NBS solution and allow the mixture to stand for 15 min at room temperature with occasional shaking. Add 1.Oml of DPPH,, mix well and dilute to volume with ethanol. Measure AA at 520 nm against a reagent blank similarly treated. 2. DC polarography, cyclic and stripping voltammetric measurements A stock solutions. 1 mh4 of ketoprofen
was prepared daily by dissolving the appropriate weight in bidistilled water and methanol (1: 1). Capsules: As previously mentioned under spectrophotometric method using doubly distilled water-methanol (1: 1) as solvent and 1 x lo-‘it4 ketoprofen. Procedure.
Use 50 ml of the supporting electrolyte solution for DC polarography and degassed with nitrogen for 20 min. For cyclic and stripping voltammetric measurements: Deaerate 10 ml of supporting electrolyte by passing nitrogen for 12 min, after an equilibrium time (15 set) record the voltammogram. The preconcentration potential is - 0.6 V and the final potential is - 1.2 V, while pulse amplitude is 50 mV and pulse repetition is 0.5 sec. Repeat the same procedure after spiking the sample solution. Perform the quantitative analysis by the standard addition method by spiking with standard solutions. RESULTS AND DISCUSSION
Spectrophotometric determination
An indirect method for the determination of ketoprofen is carried out via its reaction with NBS. The unconsumed reagent is then determined by formation of a violet colour (Lx = 520 nm) with 2,2-diphenyl-1-picryl hydrazine (DPPH,) in aqueous ethanolic medium. By subtraction from a blank, AA is proportional to the quantity of the drug. The absorption spectrum is shown in Fig. 1. The optimum conditions for the reaction between NBS (I) and 2,2-diphenyl-l-picrylhydrazine (II) were first studied. The molar ratio of I: II was found to be 1: 2 (Table 1).
641
Polarographic behaviour of Ketoprofen 0.4r
0.5r
I
I
0.1 t
wave
nm
Fig. 1. Absorption spectra of (1) ketoprofen-NBSdiphenylpicrylhydrazine, (2) NBS-diphenylpicrylhydraxine and (3) diphenylpicrylhydraxyl. Final drug concentration, 25/lgml-1.
Fig. 2. Stability time of ketoprofen with NBS and DPPH,; Final drug concentration = 25 pg ml-‘.
Polarographic behaviour The violet coloured product was stable for more than 2 h (Fig. 2) and ethanol is the best solvent (Table 2). For further studies of the reaction, an authentic sample of the violet coloured stable free radical 2,2-diphenyl- lpicrylhydrazyl in ethanol was scanned and the obtained J,_ was 520 nm, Fig. 1. So the suggested reaction mechanism is that (II) is easily oxidized by (I) to the stable radical 2,2-diphenyl-1-picrylhydrazyl (III) as shown in Scheme 2. Quantification A linear correlation (r = 0.9998) was found between AA at 520 nm and the concentration of ketoprofen in the range 5-80 pg/ml. A typical linear regression line has a slope of 0.0126 and an intercept of - 0.0099. The molar absorptivity (em=) is 3.15 x lo3 l/mol/cm. The precision (coefficient of variation) of eight replicate determinations at the 50 pug/ml level was 0.65%. This level of precision is adequate for the quality control analysis of pharmaceutical preparations. Table 1. Molar ratio of NBS and DPPH, Volume of NBS* (W
Volume of DPPH,* (ml)
0.25 0.25 0.25 0.25 0.25
0.125 0.250 0.375 0.500 0.750
*Equal concentration = 1 x IO-‘M. TAverage of three determinations.
The polarographic reduction behaviour of ketoprofen at the dropping mercury electrode was investigated in the pH range from 1 to 12 using different supporting electrolytes. Preliminary work shows that ketoprofen exhibits a well defined wave at -0.97 or - 1.35 V in both phosphate (pH 1.5-12) and nitrate media (pH 6-l 1.5), respectively. When increasing pH values the half wave potential (&) is shifted towards more negative values. An important adsorption phenomenon always accompanied the electroreduction process as it can be proven from the effect of mercury height for the reduction wave in phosphate medium at different pH values, Table 3. Ketoprofen in presence of perchlorate medium shows a well defined wave at pH 1.5 and 3 with E - 1.02 V. At higher pH values another wave appeared, and its E,,2 value lies between -0.64 and -0.97 V as seen in Fig. 3. The effect of mercury height for the two waves indicated both reduction waves are controlled by adsorption (Table 3). So, from the
Table 2. Effect of diluting solvents on the coloured product of NBS and DPPH,* Solvent
&Ot 0.151 0.299 0.459 0.619 0.618
l+Dioxane Dimethylsuphoxide Ethanol Methanol n Propanol Isopropanol
LX 520 520 520 520 520 520
A at 1_
0.575 0.517 0.617 0.551 0.578 0.582
*NBS; 0.25 ml (I x lo-‘M) per 10 ml. TDPPH,; 0.5 ml (1 x IO-‘M) per 10 ml.
KAMLAM. EMAW et al.
IIYCUOW
Scheme 2.
previous polarographic studies it was concluded that the carbonyl group is the site of the electrochemical reduction of ketoprofen and this result is in concordance with the reported one.” Stripping measurements
Preliminary investigations show that small and unusable peaks were observed in the presence of phosphate, nitrate or acetate using different pH values (l-l 1) and concentrations (0.01-0.2M). The effect of the concentration of perchlorate as supporting electrolyte, viz, 0.01, 0.02, 0.04, 0.06. 0.08, 0.1 and 0.2M and the influence of pH (2.3, 3.8, 5.5, 7.3,9 and 12) was studied. The highest signal was obtained with perchlorate (0.1 M) at pH - 3.8 and was chosen
as the ideal conditions for the adsorptive stripping measurements in this work. Cyclic voltammogram has been taken for ketoprofen, a single peak is obtained at peak potential -0.98 V and no peak is observed on scanning in the positive direction, Fig. 5. This means that the reduction product inhibits the appearance of any oxidation peak. The effect of scan rate v on the peak current or the peak potential was studied. The log ip vs log v plot is linear over the range 10-500 mV/sec with a slope of 1.2 which is in agreement with that expected for reversible reaction of surface species.” A 15 mV is negative shift in the peak potential was observed when the scan was increased in the range given.
Table 3. Influence of the supporting electrolytes on the reduction of 1 x lo-‘M ketoprofen 0. 1M phosphate
PH
-E,,z
1
-
1.5 3 3.8 5.0 5.8 6.0 6.5 7.8 9.5 10.5 11.5 12.0
0.95 1.05 0.75 0.77 0.80 0.95
Log i/log h -
1.03 1.3 1.03 1.20 1.20 1.30
-42
O.lM nitrate Log i/log h
-
1.35 1.40 -
-
0.90 1.02 -
-
42
-
0.64 0.73 0.85 0.9
0.1M perchlorate Log i/log h -E,,, _
0.94 0.97 1.02 1.03 -
1.02 1.08 1.38 1.41 1.44 1.49 -
Log i/log h 1.92
0.74 0.92 0.80 0.82 1.60 -
Polarographic behaviour of Ketoprofen
643
-L -2.2
Fig. 3. Direct current polarograms of 1 x 10e5M ketoprofen using 0.1 M perchlorate and different pH values. (A) pH 1.0; (B) pH 3.0; (C) pH 5.0; (D) pH 6.5; (E) pH 9.5; (F) pH 11.5.
20
40
60
80
loo
sThe effect of deposition potential using direct current stripping voltammetry (DCSV) on the peak current is studied over a wide range. The peak height is almost constant and maximum when the deposition potential lies between -0.6 and - 1.2 V. Figure 5 shows the peak current vs preconcentration time for l,lO, 50 and 100 x 10-8M ketoprofen, the intersections of these lines with the peak current axis may be attributed to the fact that adsorption takes place at the equilibrium time23 which was fixed at 15 sec. The break at certain stirring times means that surface coverage was attained. This is in accordance with the results obtained by cyclic voltammetric measurements since fast deposition of the molecule is observed after the first cycle.
Fig. 5. ip vs Preconcentration time using 0. I M perchlorate and pH N 3.8. (A) 1 x lo-*M; (B) 1 x lo-‘M; (C) 5 x lo-’ M and (D) 1 x low6 M ketoprofen.
Data recorded in Table 4 summarizes the characteristics of the calibration plots established with different deposition times. The linearity range from lop8 to lo-“M (0.25 x IO-* to 0.25 pg/ml) ketoprofen indicates strong adsorption behaviour of the drug for all of the deposition times applied. Under these conditions the charge transferred for the reduction step corresponds to 24.4 x lo-’ C as calculated by integration of the area under the peak.24 A monolayer surface coverage of 9.04 x lo-‘O mol/cm* can be
I
loonA
t
0.6
I
I
I
0.8
I
1.2 -B, v VI. Ag/Ag&cl(,,
Fig. 4. Multicyclic voltamperograms for 1 x 10e5 M ketoprofen in presence of 0.1 M perchlorate pH 3.8 and scan rate lOOm/sec., Curve l-first cycle, curve 2, 3, . . . are successive scans at the same drop.
644
KAMLA
M. EMARAet al.
Table 4. Characteristics of ketoprofen calibration plots using O.lM perchlorate at pH 3.8
Dep. Time (s)
Linearity range
Equation
Correlation coefficient
0 10 20 30
10-s-10lo-*-lo10-*-10-’ lo-*-lo-’
Y=16x+6 Y = 13.3X + 13 Y=2O.OX+18 Y = 22.2X + 32
0.997 0.996 0.998 0.998
*Yin nA, X in 10-‘1I4, slope in nA/10-8, intercept in nA.
estimated by dividing the charge by nFA (n = 2 electrons).” Consequently, adsorbed ketoprofen molecule occupies an area of 0.184 nm’. The detection limit of 2 x 10e9M (0.508 ng/ml) ketoprofen was determined using 20 second preconcentration time by DCSV. Dosage forms
The proposed methods were applied for the determination of ketoprofen in powder form and in capsules at room temperature (23 & 2°C). The results obtained by both methods are in good accordance with the reported and official assay methods as revealed by statistical analysis (see Tables 5 and 6). The result of the analysis Table 5. Assay of ketoprofen powder using the suggested and reported spectrophotometric methods Amount of drug (rra) 10 20 30 40 50 60 Average recovery (%) *SD
Recovery* (%) Suggested Reported method method+ 98.71 99.33 96.64 100.09 101.63 98.22
101.65 97.32 97.48 99.11 98.35 100.72
99.1 + 1.70
99.10 * 1.97
Table 7. Determination of ketoprofen capsules by the proposed and official methods
Drug form Capsules#
1. J. E. F. Reynolds, Editor, Martindale, The Extra Pharmacopoeia, 28th Edition, Pharmaceutical Press, London, 1982, p. 261. 2. R. N. Brogden, T. M. Speight and G. C. Avery, Drugs, 1974, 8, 168. 3. British Pharmacopoeia,
5. 6. 7.
98.66 101.72 98.44 97.94 100.86 99.55 99.5 + 1.49
-’ _
99.32 102.01 100.34 97.38 101.14 99.22 99.9 + 1.63
*Average of three experiments. TReference (8). SProfenid capsules (Alexandria Pharm. Co., Egypt under licence of Phone Pulence, Paris-France) labelled to contain 50 mg ketoprofen per capsule.
100.0 f 0.92
REFERENCES
Table 6. Application of the voltammetric and reported DOlarOaraDhiCmethods for the assay of nrofenid ca~sulest
1 2 3 4 5 6 Average Recovery (%) *SD
101.9 f 1.04 F = 1.28 f = 0.68
recorded in Table 7 indicate the suitability of both methods to the assay of the drug in capsules. The calculated values of F- and t(at 95% confidence level) did not exceed the tabulated (theoretical) ones. This means that there is no significant difference between the proposed and official methods with respect to precision and accuracy. The proposed adsorptive stripping voltammetry (DCSV) can be used successfully for determination of ketoprofen concentration in profenid capsules. In addition, the great sensitivity of DCSV method allows the quantitative estimation of the drug in biological media which will be the subject of further work.
4.
Recovery* (%) Voltammetric Polarographic method method
99.5 f 0.89 §F = 1.07 I = 0.57
*Average of five determinations. tRef. 3. SProfenid capsules (Alexandria Phann. Co., Egypt under licence of Phone Poulence, Paris-France) labelled to contain 50 mg ketoprofen per capsule. §Tabulated t (n = 5), P 0.05 = 2.57. Tabulated F(5,5), P 0.05 = 5.05.
*Average of three experiments. tRef. (6).
Amount of drug (M x 10-7)
Recovery (%) f SD* Voltammetric Calorimetric Official method method method?
8.
Her Majesty’s Stationery Office, London, 1988, pp. 325 and 639. N. Blazevic, M. Zinic, T. Kovac, V. Sunjic and F. Kajfex, Acta. Pharm. Jugosl., 1975, 25, 155. B. Lotti, Boll. Chim. Farm., 1975, 114, 351. B. Unterhalt, Pharm. Ztg., 1978, 123, 1801. C. S. P. Sastry, A. S. R. P. Tipirneni and M. V. Suryanarayana, Analyst, 1989, 114, 513. T. Guneri and L. Kirilmaz, Acta Pharm. Turc., 1988,30,
149. 9. M. H. Abdel-Hay, M. A. Korany, M. M. Bedair and A. A. Gazy, Anal. L&t., 1990, 23, 281. 10. A. Lawrence and G. Leslie, Analyst 1984, 109, 57.
11. G. Kanoute, P. Boucly, E. Guemet-Nivaud and M. Guernet, J. Ann. Pharma. Fr., 1985, 43, 265. 12. B. Normand, J. Lefrancois, H. Ong and G. Edward, J. Assoc. Off. Anal. Chem., 1989, 72, 559. 13. S. C. Chi and H. W. Jun, J. Liq. Chromatogr., 1989, 12, 2931. 14. B. B. Lott, Chin Farm., 1975, 114, 351. 15. P. Populaire, B. Terlain, S. Pascal, B. Decouvelaere, G. Lebreton, A. Renard and P. Thomas, Ann. Pharm. Fr., 1973, 31, 679.
Polarographic behaviour of Ketoprofen 16. C. M. Moore and I. R. Tebbett, Forensic Sci. Ink, 1987, 34, 155. 17. R. A. Upton, J. N. Buskin, T. W. Guentert, R. L. Williams and S. Riegelman, J. Chromntogr., 1980, 190, 119. 18. T. M. Jefferies, W. 0. A. Thomas and R. T. Par&t, J. Chromatogr., 1979, 162, 122. 19. J. Wang, Stripping Analysis. VCH Publishers, Deerfield Beach, Fl. 1985. 20. N. Abou El-Maali, A. M. M. Ah, M. Khodari and
21. 22. 23. 24.
645
M. A. Ghandour, Bioelectrochemistry and Bioenergetics, 1991, 26,485. A. M. M. Ah, N. Abo El-Maali and M. A. Ghandour, Electroanalysis, 1994, 4, in press. J. Wang, P. Tuzhi, M. S. Lin and T. Tapia, Talanta, 1986, 33, 707. P. Delahay and C. Fike, J. Am. Chem. Sot., 1958, &IO, 2628. R. C. Gut-ha and L. D. Bowers, J. Electroanal. Chem., 1983, 189, 146.
Copyright 0 1994Elscvicr Science Ltd Printed in GreatBritain. All rightsresewed 0039-9140/94$7.00+ 0.00
Pergamon
THE POLAROGRAPHIC BEHAVIOUR OF KETOPROFEN AND ASSAY OF ITS CAPSULES USING SPECTROPHOTOMETRIC AND VOLTAMMETRIC METHODS KAMLA M. EMARA,* Azw
M. M. ALI
and NAGWA ABG-EL MAALI
Department of Analytical Pharmaceutical Chemistry, Faculty of Pharmacy and Department of Chemistry, Faculty of Science, University of Assiut, As&t, Egypt (Received 15 December 1992. Revised 21 September 1993. Accepted 21 September 1993) Summary-The quantitative determination of ketoprofen using spectrophotometric and voltammetric methods are described. The spectrophotometric procedure depends upon the reaction of ketoprofen with N-bromosuccinimide (NBS). The residual reagent is then determined by formation of violet colour with 2,2-diphenyl-1-picryl hydrazine (DPPH,). The consumed NBS would correspond to ketoprofen. Beer’s law is valid over the concentration range 5-80 rg/ml of the drug. Direct current (DC) polarography allows to study the reduction behaviour of ketoprofen at the dropping mercury electrode @ME) using different supporting electrolytes at different pH values. Direct current stripping voltammetry (DCSV) was used for the quantitative measurements of the drug. The calibration graph of peak current vs concentration was linear from 0.254 x lo-* to 0.254 &ml. In model solutions as little as 5.08 x low4 ng/ml ketoprofen can be detected by DCSV. Both methods were applied successfully for the determination of ketoprofen either in pure or dosage forms.
Ketoprofen 2-(3-benzoylphenyl) propionic acid (I), is an anti-inflammatory and analgesic drug. It is used in the treatment of rheumatoid arthritis and osteoarthritis.‘** cH3 I
Scheme 1.
The compendia1 method for the determination of authentic drug involves titration with standard sodium hydroxide in aqueous ethanolic medium, while ketoprofen in capsules has been determined by U.V. spectrophotometry at 258 nm after extraction with methanol (75%).3 Existing analytical procedures for the assay of ketoprofen include potentiometric titration,4 ultraviolet spectrophotometry,“5 visible spectrophotometry,“’ polarography,%’ gas or liquid chromatography4*‘0 and high-performance liquid chromatography. ‘I Reported methods for its *Author for correspondence.
determination in biological samples include spectrophotometry,‘* calorimetry or polarography,i3 thin-layer or paper chromatography” and high-performance liquid chromatography.“6 Although the nitrophenylhydrazine method9 and the coloured ion pairs (with basic dyes) methodsa yielded comparable results, the spectrophotometric method proposed was found to be preferable for the analysis of capsules because it was not as tedious as the nitrophenylhydrazine method and does not require extraction prior to spectrophotometric measurements. Moreover, the method is more convenient than the B.P. (1988) method for the assay of ketoprofen powder as only a small amount of the drug is necessary for each analysis. Stripping voltammetry is an important technique for trace determination of many inorganic and organic substances.lg The adsorptive strip ping technique has been used successfully for determination of subnanogram level of several drugs.20v2’ The present work deals with the quantitative determination of ketoprofen using direct current stripping voltammetry as well as spectrophotometric methods. In addition the polarographic behaviour of the drug was investigated. Both methods are simple, rapid, sensitive, reproducible and easy to apply to routine usage. 639
640
KAMLA
M. EMARAet al.
EXPERIMENTAL
Materials and reagents
Ketoprofen: supplied by Alexandria Pharm. Co. Alexandria, Egypt. NBS (SIGMA, U.S.A.) 1 x 10-3M (0.178 mg/ml) in distilled water. 2,2 - Diphenyl - 1 - picrylhydrazine (DPPH,) (SIGMA, U.S.A.), 1 x 10m3M (0.395 mg/ml) was freshly prepared in ethanol. 2,2 - Diphenyl - 1 - picrylhydrazyl (SIGMA, U.S.A.), 1 x 10p3M (0.394 mg/ml) was freshly prepared in ethanol. Supporting electrolytes: 0.1 M 0.1 M l O.lM l O.lM ment lytes.
l l
l
0.1N
perchloric acid. phosphoric acid. nitric acid. sodium hydroxide; used for adjustof the pH of the supporting electroacetic acid-sodium
acetate buffer.
All chemicals and solvents were of analytical grade. Instrumentation
Uvidec-320 spectrophotometer (JASCO, Tokyo, Japan). l Polarograph PRG 5 (Tacussel, France) equipped with three electrode potentiostatic system. Dropping mercury electrode @ME) has the capillary characteristics m = 2.10 mg/sec, t = 4.11 set (open circuit) and m 2/3t‘I6= 2.07 mg2/3/sec”2. l For voltammetric studies: an EG & G Princeton Applied Research Corp. (PAR) Model 264A was used, coupled with a PAR 303A Static Mercury Drop Electrode (SMDE) (drop size: medium, area of the drop: 0.014 cm*). The polarographic cell (PAR Model KOO60) was fitted with an Ag/AgCl saturated KCl, reference electrode and a platinum wire counter electrode. A PAR 305 stirrer was connected to the 303A SMDE and a PAR Model REOOS9 X-Y recorder was used for the collection of the experimental data. l
Method I. Spectrophotometric method Preparation of sample solutions Ketoprofen powder. Weigh accurately 25 mg of
ketoprofen and dissolve in 100 ml ethanol. Use 2-ml aliquot of this solution for the procedure. Capsules. Shake a quantity of the mixed con-
tents of 20 capsules containing 25 mg of ketoprofen for 10 min with 50 ml ethanol. Filter and wash the residue with sufficient ethanol to give a volume of 100 ml. Use 2 ml aliquot of this solution for the procedure. Procedure.
Pipette 2.0 ml of the sample solution into a 10 ml calibrated flask. Add 0.5 ml of NBS solution and allow the mixture to stand for 15 min at room temperature with occasional shaking. Add 1.Oml of DPPH,, mix well and dilute to volume with ethanol. Measure AA at 520 nm against a reagent blank similarly treated. 2. DC polarography, cyclic and stripping voltammetric measurements A stock solutions. 1 mh4 of ketoprofen
was prepared daily by dissolving the appropriate weight in bidistilled water and methanol (1: 1). Capsules: As previously mentioned under spectrophotometric method using doubly distilled water-methanol (1: 1) as solvent and 1 x lo-‘it4 ketoprofen. Procedure.
Use 50 ml of the supporting electrolyte solution for DC polarography and degassed with nitrogen for 20 min. For cyclic and stripping voltammetric measurements: Deaerate 10 ml of supporting electrolyte by passing nitrogen for 12 min, after an equilibrium time (15 set) record the voltammogram. The preconcentration potential is - 0.6 V and the final potential is - 1.2 V, while pulse amplitude is 50 mV and pulse repetition is 0.5 sec. Repeat the same procedure after spiking the sample solution. Perform the quantitative analysis by the standard addition method by spiking with standard solutions. RESULTS AND DISCUSSION
Spectrophotometric determination
An indirect method for the determination of ketoprofen is carried out via its reaction with NBS. The unconsumed reagent is then determined by formation of a violet colour (Lx = 520 nm) with 2,2-diphenyl-1-picryl hydrazine (DPPH,) in aqueous ethanolic medium. By subtraction from a blank, AA is proportional to the quantity of the drug. The absorption spectrum is shown in Fig. 1. The optimum conditions for the reaction between NBS (I) and 2,2-diphenyl-l-picrylhydrazine (II) were first studied. The molar ratio of I: II was found to be 1: 2 (Table 1).
641
Polarographic behaviour of Ketoprofen 0.4r
0.5r
I
I
0.1 t
wave
nm
Fig. 1. Absorption spectra of (1) ketoprofen-NBSdiphenylpicrylhydrazine, (2) NBS-diphenylpicrylhydraxine and (3) diphenylpicrylhydraxyl. Final drug concentration, 25/lgml-1.
Fig. 2. Stability time of ketoprofen with NBS and DPPH,; Final drug concentration = 25 pg ml-‘.
Polarographic behaviour The violet coloured product was stable for more than 2 h (Fig. 2) and ethanol is the best solvent (Table 2). For further studies of the reaction, an authentic sample of the violet coloured stable free radical 2,2-diphenyl- lpicrylhydrazyl in ethanol was scanned and the obtained J,_ was 520 nm, Fig. 1. So the suggested reaction mechanism is that (II) is easily oxidized by (I) to the stable radical 2,2-diphenyl-1-picrylhydrazyl (III) as shown in Scheme 2. Quantification A linear correlation (r = 0.9998) was found between AA at 520 nm and the concentration of ketoprofen in the range 5-80 pg/ml. A typical linear regression line has a slope of 0.0126 and an intercept of - 0.0099. The molar absorptivity (em=) is 3.15 x lo3 l/mol/cm. The precision (coefficient of variation) of eight replicate determinations at the 50 pug/ml level was 0.65%. This level of precision is adequate for the quality control analysis of pharmaceutical preparations. Table 1. Molar ratio of NBS and DPPH, Volume of NBS* (W
Volume of DPPH,* (ml)
0.25 0.25 0.25 0.25 0.25
0.125 0.250 0.375 0.500 0.750
*Equal concentration = 1 x IO-‘M. TAverage of three determinations.
The polarographic reduction behaviour of ketoprofen at the dropping mercury electrode was investigated in the pH range from 1 to 12 using different supporting electrolytes. Preliminary work shows that ketoprofen exhibits a well defined wave at -0.97 or - 1.35 V in both phosphate (pH 1.5-12) and nitrate media (pH 6-l 1.5), respectively. When increasing pH values the half wave potential (&) is shifted towards more negative values. An important adsorption phenomenon always accompanied the electroreduction process as it can be proven from the effect of mercury height for the reduction wave in phosphate medium at different pH values, Table 3. Ketoprofen in presence of perchlorate medium shows a well defined wave at pH 1.5 and 3 with E - 1.02 V. At higher pH values another wave appeared, and its E,,2 value lies between -0.64 and -0.97 V as seen in Fig. 3. The effect of mercury height for the two waves indicated both reduction waves are controlled by adsorption (Table 3). So, from the
Table 2. Effect of diluting solvents on the coloured product of NBS and DPPH,* Solvent
&Ot 0.151 0.299 0.459 0.619 0.618
l+Dioxane Dimethylsuphoxide Ethanol Methanol n Propanol Isopropanol
LX 520 520 520 520 520 520
A at 1_
0.575 0.517 0.617 0.551 0.578 0.582
*NBS; 0.25 ml (I x lo-‘M) per 10 ml. TDPPH,; 0.5 ml (1 x IO-‘M) per 10 ml.
KAMLAM. EMAW et al.
IIYCUOW
Scheme 2.
previous polarographic studies it was concluded that the carbonyl group is the site of the electrochemical reduction of ketoprofen and this result is in concordance with the reported one.” Stripping measurements
Preliminary investigations show that small and unusable peaks were observed in the presence of phosphate, nitrate or acetate using different pH values (l-l 1) and concentrations (0.01-0.2M). The effect of the concentration of perchlorate as supporting electrolyte, viz, 0.01, 0.02, 0.04, 0.06. 0.08, 0.1 and 0.2M and the influence of pH (2.3, 3.8, 5.5, 7.3,9 and 12) was studied. The highest signal was obtained with perchlorate (0.1 M) at pH - 3.8 and was chosen
as the ideal conditions for the adsorptive stripping measurements in this work. Cyclic voltammogram has been taken for ketoprofen, a single peak is obtained at peak potential -0.98 V and no peak is observed on scanning in the positive direction, Fig. 5. This means that the reduction product inhibits the appearance of any oxidation peak. The effect of scan rate v on the peak current or the peak potential was studied. The log ip vs log v plot is linear over the range 10-500 mV/sec with a slope of 1.2 which is in agreement with that expected for reversible reaction of surface species.” A 15 mV is negative shift in the peak potential was observed when the scan was increased in the range given.
Table 3. Influence of the supporting electrolytes on the reduction of 1 x lo-‘M ketoprofen 0. 1M phosphate
PH
-E,,z
1
-
1.5 3 3.8 5.0 5.8 6.0 6.5 7.8 9.5 10.5 11.5 12.0
0.95 1.05 0.75 0.77 0.80 0.95
Log i/log h -
1.03 1.3 1.03 1.20 1.20 1.30
-42
O.lM nitrate Log i/log h
-
1.35 1.40 -
-
0.90 1.02 -
-
42
-
0.64 0.73 0.85 0.9
0.1M perchlorate Log i/log h -E,,, _
0.94 0.97 1.02 1.03 -
1.02 1.08 1.38 1.41 1.44 1.49 -
Log i/log h 1.92
0.74 0.92 0.80 0.82 1.60 -
Polarographic behaviour of Ketoprofen
643
-L -2.2
Fig. 3. Direct current polarograms of 1 x 10e5M ketoprofen using 0.1 M perchlorate and different pH values. (A) pH 1.0; (B) pH 3.0; (C) pH 5.0; (D) pH 6.5; (E) pH 9.5; (F) pH 11.5.
20
40
60
80
loo
sThe effect of deposition potential using direct current stripping voltammetry (DCSV) on the peak current is studied over a wide range. The peak height is almost constant and maximum when the deposition potential lies between -0.6 and - 1.2 V. Figure 5 shows the peak current vs preconcentration time for l,lO, 50 and 100 x 10-8M ketoprofen, the intersections of these lines with the peak current axis may be attributed to the fact that adsorption takes place at the equilibrium time23 which was fixed at 15 sec. The break at certain stirring times means that surface coverage was attained. This is in accordance with the results obtained by cyclic voltammetric measurements since fast deposition of the molecule is observed after the first cycle.
Fig. 5. ip vs Preconcentration time using 0. I M perchlorate and pH N 3.8. (A) 1 x lo-*M; (B) 1 x lo-‘M; (C) 5 x lo-’ M and (D) 1 x low6 M ketoprofen.
Data recorded in Table 4 summarizes the characteristics of the calibration plots established with different deposition times. The linearity range from lop8 to lo-“M (0.25 x IO-* to 0.25 pg/ml) ketoprofen indicates strong adsorption behaviour of the drug for all of the deposition times applied. Under these conditions the charge transferred for the reduction step corresponds to 24.4 x lo-’ C as calculated by integration of the area under the peak.24 A monolayer surface coverage of 9.04 x lo-‘O mol/cm* can be
I
loonA
t
0.6
I
I
I
0.8
I
1.2 -B, v VI. Ag/Ag&cl(,,
Fig. 4. Multicyclic voltamperograms for 1 x 10e5 M ketoprofen in presence of 0.1 M perchlorate pH 3.8 and scan rate lOOm/sec., Curve l-first cycle, curve 2, 3, . . . are successive scans at the same drop.
644
KAMLA
M. EMARAet al.
Table 4. Characteristics of ketoprofen calibration plots using O.lM perchlorate at pH 3.8
Dep. Time (s)
Linearity range
Equation
Correlation coefficient
0 10 20 30
10-s-10lo-*-lo10-*-10-’ lo-*-lo-’
Y=16x+6 Y = 13.3X + 13 Y=2O.OX+18 Y = 22.2X + 32
0.997 0.996 0.998 0.998
*Yin nA, X in 10-‘1I4, slope in nA/10-8, intercept in nA.
estimated by dividing the charge by nFA (n = 2 electrons).” Consequently, adsorbed ketoprofen molecule occupies an area of 0.184 nm’. The detection limit of 2 x 10e9M (0.508 ng/ml) ketoprofen was determined using 20 second preconcentration time by DCSV. Dosage forms
The proposed methods were applied for the determination of ketoprofen in powder form and in capsules at room temperature (23 & 2°C). The results obtained by both methods are in good accordance with the reported and official assay methods as revealed by statistical analysis (see Tables 5 and 6). The result of the analysis Table 5. Assay of ketoprofen powder using the suggested and reported spectrophotometric methods Amount of drug (rra) 10 20 30 40 50 60 Average recovery (%) *SD
Recovery* (%) Suggested Reported method method+ 98.71 99.33 96.64 100.09 101.63 98.22
101.65 97.32 97.48 99.11 98.35 100.72
99.1 + 1.70
99.10 * 1.97
Table 7. Determination of ketoprofen capsules by the proposed and official methods
Drug form Capsules#
1. J. E. F. Reynolds, Editor, Martindale, The Extra Pharmacopoeia, 28th Edition, Pharmaceutical Press, London, 1982, p. 261. 2. R. N. Brogden, T. M. Speight and G. C. Avery, Drugs, 1974, 8, 168. 3. British Pharmacopoeia,
5. 6. 7.
98.66 101.72 98.44 97.94 100.86 99.55 99.5 + 1.49
-’ _
99.32 102.01 100.34 97.38 101.14 99.22 99.9 + 1.63
*Average of three experiments. TReference (8). SProfenid capsules (Alexandria Pharm. Co., Egypt under licence of Phone Pulence, Paris-France) labelled to contain 50 mg ketoprofen per capsule.
100.0 f 0.92
REFERENCES
Table 6. Application of the voltammetric and reported DOlarOaraDhiCmethods for the assay of nrofenid ca~sulest
1 2 3 4 5 6 Average Recovery (%) *SD
101.9 f 1.04 F = 1.28 f = 0.68
recorded in Table 7 indicate the suitability of both methods to the assay of the drug in capsules. The calculated values of F- and t(at 95% confidence level) did not exceed the tabulated (theoretical) ones. This means that there is no significant difference between the proposed and official methods with respect to precision and accuracy. The proposed adsorptive stripping voltammetry (DCSV) can be used successfully for determination of ketoprofen concentration in profenid capsules. In addition, the great sensitivity of DCSV method allows the quantitative estimation of the drug in biological media which will be the subject of further work.
4.
Recovery* (%) Voltammetric Polarographic method method
99.5 f 0.89 §F = 1.07 I = 0.57
*Average of five determinations. tRef. 3. SProfenid capsules (Alexandria Phann. Co., Egypt under licence of Phone Poulence, Paris-France) labelled to contain 50 mg ketoprofen per capsule. §Tabulated t (n = 5), P 0.05 = 2.57. Tabulated F(5,5), P 0.05 = 5.05.
*Average of three experiments. tRef. (6).
Amount of drug (M x 10-7)
Recovery (%) f SD* Voltammetric Calorimetric Official method method method?
8.
Her Majesty’s Stationery Office, London, 1988, pp. 325 and 639. N. Blazevic, M. Zinic, T. Kovac, V. Sunjic and F. Kajfex, Acta. Pharm. Jugosl., 1975, 25, 155. B. Lotti, Boll. Chim. Farm., 1975, 114, 351. B. Unterhalt, Pharm. Ztg., 1978, 123, 1801. C. S. P. Sastry, A. S. R. P. Tipirneni and M. V. Suryanarayana, Analyst, 1989, 114, 513. T. Guneri and L. Kirilmaz, Acta Pharm. Turc., 1988,30,
149. 9. M. H. Abdel-Hay, M. A. Korany, M. M. Bedair and A. A. Gazy, Anal. L&t., 1990, 23, 281. 10. A. Lawrence and G. Leslie, Analyst 1984, 109, 57.
11. G. Kanoute, P. Boucly, E. Guemet-Nivaud and M. Guernet, J. Ann. Pharma. Fr., 1985, 43, 265. 12. B. Normand, J. Lefrancois, H. Ong and G. Edward, J. Assoc. Off. Anal. Chem., 1989, 72, 559. 13. S. C. Chi and H. W. Jun, J. Liq. Chromatogr., 1989, 12, 2931. 14. B. B. Lott, Chin Farm., 1975, 114, 351. 15. P. Populaire, B. Terlain, S. Pascal, B. Decouvelaere, G. Lebreton, A. Renard and P. Thomas, Ann. Pharm. Fr., 1973, 31, 679.
Polarographic behaviour of Ketoprofen 16. C. M. Moore and I. R. Tebbett, Forensic Sci. Ink, 1987, 34, 155. 17. R. A. Upton, J. N. Buskin, T. W. Guentert, R. L. Williams and S. Riegelman, J. Chromntogr., 1980, 190, 119. 18. T. M. Jefferies, W. 0. A. Thomas and R. T. Par&t, J. Chromatogr., 1979, 162, 122. 19. J. Wang, Stripping Analysis. VCH Publishers, Deerfield Beach, Fl. 1985. 20. N. Abou El-Maali, A. M. M. Ah, M. Khodari and
21. 22. 23. 24.
645
M. A. Ghandour, Bioelectrochemistry and Bioenergetics, 1991, 26,485. A. M. M. Ah, N. Abo El-Maali and M. A. Ghandour, Electroanalysis, 1994, 4, in press. J. Wang, P. Tuzhi, M. S. Lin and T. Tapia, Talanta, 1986, 33, 707. P. Delahay and C. Fike, J. Am. Chem. Sot., 1958, &IO, 2628. R. C. Gut-ha and L. D. Bowers, J. Electroanal. Chem., 1983, 189, 146.