Kinetic Estimation of Ibuprofen and Nimesulide in Pharmaceuticals

 

B.S. Virupaxappa1*, K.H. Shivaprasad1, Raviraj M. Kulkarni2 and M.S. Latha3

1Department of Chemistry, PG Centre, Bellary, Gulbarga University, Gulbarga, Karnataka, (INDIA)

2Department of Chemistry, Gogte Institute of Technology, Udyambag, Belgaum, Karnataka, (INDIA)

3Department of Chemistry, G M Institute of Technology Davangere, Karnataka, (INDIA)

*Corresponding Author E-mail: lathamschem97@gmail.com

 

ABSTRACT:

Simple Kinetic Spectrophotometric methods have been developed and validated for the estimation of Anti inflammatory drugs, namely Ibuprofen and Nimesulide in pharmaceutical dosage forms. The method is based on the oxidation of Ibuprofen and Nimesulide with alkaline potassium permanganate. In alkaline medium permanganate (Mn+7) oxidizes Ibuprofen and Nimesulide undergoes one electron reduction to give a green colored manganate ions (Mn+6) which has wavelength maximum at 610 nm, and unreacted permanganate at 526 nm. The Fixed time (at 240 sec) and Rate constant method were utilized for constructing the calibration graphs. The calibration graphs are linear in the concentration ranges from 30.8–308 μgmL−1 and 20.6 – 206 μgmL−1 using the Fixed time methods and Rate constant methods respectively. The results are validated statistically and checked through recovery studies. The method has been successfully applied for the determination of the studied Anti inflammatory drugs in commercial dosage forms.

 

KEYWORDS: Ibuprofen, Nimesulide, Kinetic spectrophotometry, Fixed time method, Rate constant method.

 


 

1. INTRODUCTION:

Nimesulide, chemically N-(4-nitro-2-phenoxyphenyl) methanesulphonamide (Fig-1) is a relatively new non-steroidal anti inflammatory, antipyretic and analgesic drug1.Nimesulide is a potent anti inflammatory agent which has a reduced gastric irritancy. The drug is receiving much attention these days due to its significant Selectivity towards cyclooxygenase-2 (COX-2) versus COX-1 inhibition. It is a novel compound in the respect that it exhibits an acidic character that is attributed to sulfonanilide rather to a carboxyl group, which is the case with all other acidic anti inflammatory agents. A survey of literature reveals that there are a very few methods available for the determination of Nimesulide .The methods includes voltammetry2, HPLC3, Fluorimetry4, Calorimetry5, and spectrophotometry6-15.One of the spectrophotometric methods which is already reported14,15 makes use of KMnO4 and organic dye has certain serious limitations. Other spectrophotometric methods suffer from disadvantages like extraction, long time required for the reaction to complete, narrow range of determination and lack of sensitivity. In continuation of our work on the spectrophtometric determination of pharmaceutically important compounds1619.

 

Ibuprofen [(±)-2-(p-ionic acidsobutylphenyl) propionic acid] (Fig-1) is a non steroidal anti inflammatory drugs with good analgetic, anti-inflammatory and antipyretic effects. Many techniques and procedures have been published for the quantitative determination of Ibuprofen. These procedures include non aqueous titration20, polarography21, and colorimetric titration22, and differential scanning calorimetry23, first and second order derivative UV spectrophotometry24,25, colourimetry26,27, IR spectrophotometry28, and H NMR spectrophotometry29.  In addition chromatographic procedures are also employed using TLC30, GC31, and HPLC32-34. According to the BP 1988 ibuprofen and its tablets are determined by an acid base titration. The present paper reports elegant methods for the kinetic spectrophotometric determination of Ibuprofen and Nimesulide using alkaline KMnO4 as a new reagent.

 

A)     Ibuprofen                                                    B) Nimesulide

 

Fig 1. Structures of studied Anti Inflammatory drugs.

2 EXPERIMENTAL:

2.1 Apparatus:

A Peltier Accessory (Temperature controlled) Varian Cary 50 model UV-Vis Spectrophotometer equipped with 10 mm quartz cell was used for all spectral measurements. Systronics pH meter were used for the accurate pH determinations.

 

2.2 Materials and reagents:

All the materials were of analytical reagent grade, and the solutions were prepared with double distilled water, samples of Ibuprofen and Nimesulide were generously supplied by their respective manufactures and were used without further purification. The Ibuprofen and Nimesulide were procured as gift samples from (Recon Health care, Bengaluru, Karnataka) and (Torrent Research center, Ahmedabad). Potassium permanganate (Merck, Germany) 1 x 10-3 M solution was prepared by dissolving 0.0395g KMnO4 in 100 ml of double distilled water, followed by boiling and filtration through sintered glass. Potassium permanganate solution should be freshly prepared and its molarity was checked titrimetrically. Sodium hydroxide (Merck, Germany), 2M NaOH was prepared by dissolving 8g of NaOH in 100ml of double distilled water. 2 M perchloric acid was prepared by dissolving 17.5 ml of HClO4 in 100 ml of double distilled water. 2M NaClO4: was prepared by dissolving equal proportions of 2M NaOH and 2M HClO4.

 

2.3 Pharmaceutical formulations:

The following available commercial preparations were analyzed Tablets such as Brufen, Ibugin, Ibupal ,Ibrumac, capsules like Ibubid and Ibugesic containing Ibuprofen and tablets like Antipen, Nicip, Nimulide, Nise and syrup like Cumin and Relisulide containing Nimisulide are used for the determination.

 

2.4 Preparations of standard solution:

A Working standard solution of 0.01 M Ibuprofen was prepared by dissolving 0.208 g in 100 ml of 0.1 N NaOH and Nimesulide was prepared by dissolving in 0.308 g in 100 ml of 0.1 N NaOH.

 

2.5 Kinetic procedure for Anti Inflammatory drugs determination:

All kinetic measurements were performed under pseudo first order conditions where Ibuprofen and Nimesulide used were at least 10 fold excess over permanganate at a constant ionic strength of 0.4 mol dm-3.The reaction was initiated by mixing previously thermostatted solutions of KMnO4 and anti inflammatory drugs Ibuprofen and Nimesulide, which also contained the required quantities of HClO4 and NaClO4 to maintain the required acidity and ionic strength respectively. The temperature maintained at 25 ±0.1 .c. The course of the reaction was followed by monitoring the decrease in the absorbance of KMnO4 at 526nm for both Ibuprofen and Nimesulide in alkaline medium and also increase in the absorbance of manganate ion (Mn+6) at 610nm was monitored for Ibuprofen and Nimesulide oxidation in alkaline medium.

2.6. 1 Procedure for Ibuprofen Tablets and capsules:

20 Tablets were accurately weighed and powdered in a mortar. An amount of the tablet mass equivalent to one tablet was dissolved in about 30 ml of acetone was added and the mixture was shaken for 5 min. The mixture was filtered using Whatman No. 42 filter paper and the filtrate was evaporated to dryness on a water bath. The residue was washed thoroughly several times with water before dissolving it in 0.1 N NaOH. The solution was then transferred into a 50 ml volumetric flask, made up to the mark with 0.1 N NaOH and suitable aliquot was then subjected to analysis using the procedure described under method 2.5 after diluting to 0.01 M    solution.

 

The contents of 10 capsules were evacuated and well mixed. Then an accurately weighed amount equivalent to 0.208 gm evacuated capsules of each drug transferred into a 100 mL beaker, and then the procedure was continued as described under tablets after diluting to 0.01 M Solutions.

 

2.6. 2 Procedure for Nimesulide Tablets and syrups:

20 Tablets were powdered and mixed thoroughly an amount equivalent to 50 mg of Nimesulide is dissolved in about 30 ml of acetone was added and the mixture was shaken for 5 min. The mixture was filtered using Whatman No. 42 filter paper and the filtrate was evaporated to dryness on a water bath. The residue was washed thoroughly several times with water before dissolving it in 0.01 M NaOH Solution. The solution was then transferred into a 50 ml volumetric flask, made up to the mark with  0.1 M NaOH and suitable aliquot of this solution was treated as described above for pure sample after diluting to 0.01 M    solution.

For the syrup, an appropriate volume of the sample was analyzed for Nimesulide using the procedure described for a pure sample.

 

3 .RESULT AND DISCUSSIONS:

Potassium permanganate as strong oxidizing agent has been used in oxidimetric analytical method for determination of many compounds. During the course of the reaction, the valence of manganese changes. The heptavalent manganese ion changes to the green color (Mn VI), while in neutral and acid medium, the permanganate is reduced to color less (Mn II). The behavior of permanganate was the basis for its uses in its development of spectrophotometric method. The absorption spectrum of aqueous potassium permanganate solution in alkaline medium exhibited an absorption band at 526 nm. The additions of any of the studied drugs to this solution produce a new characteristic band at 610 nm. This band is due to the formation of manganate ion, which resulted from the oxidation of anti inflammatory drugs by potassium permanganate in alkaline medium. The intensity of the color increases with time; therefore a kinetically based method was developed for the determination of anti inflammatory drugs in their pharmaceutical dosage formulations. The different variables that affect the formation of manganate ion were studied and optimized. Calibration graph of various kinetic procedures are given below.

Kinetic Procedure of Anti inflammatory drugs:

The rate constant, Fixed time methods and Fixed concentration method were used for determining Ibuprofen and Nimesulide, and the best method was choosen based on applicability, the slope of the calibration graph, the intercept and the Correlation coefficient (r).

 

Rate constant method:

Pseudo-first order rate constants were calculated for Ibuprofen and Nimesulide Concentrations in the range from 30.8–308 μgmL−1 and 20.6 – 206 μgmL−1 and are presented in Table 3 and 4 respectively. A plot of Kobs versus [Ibuprofen and Nimesulide] is drawn, which was used as a calibration graph (Fig 2 and 3). The following equation was obtained.

Kobs = 0.1886 x + 0.0007[Ibuprofen]    (r= 0.994)

Kobs = 0.6086 x + 0.0005 [Ibuprofen]   (r= 0.992)

Kobs = -0.005x + 0.0027   [Nimesulide]    (r=0.991)

Kobs = 0.075x + 0.002      [Nimesulide]     (r=0.990)

 

Fig 2. Calibration graph of Ibuprofen for  Rate Constant method.

 

Fig 3. Calibration graph of Nimisulide for  Rate constant method.

 

Fixed time method:

A pre-selected time (120secs) was fixed and the absorbance was measured for different concentrations of drugs (Table 1). A plot of the absorbance versus the initial concentration of Anti inflammatory drugs was drawn, which was linear and could be used as a calibration graph (Fig 4 and 5).This led to the following equation:

 

Absorbance = -0.0039x + 0.0026 [Ibuprofen] (r= 0.990)

Absorbance = 0.0091 x -0.0029 [Ibuprofen]   (r=0.983)

Absorbance = -0.0052 x + 0.0027 [Nimesulide](r=0.991)

Absorbance = 0.0075x + 0.0023 [Nimesulide] (r=0.990).

 

The range of the drug concentrations giving the most acceptable calibration graph with the above was 30.2- 302 µg/ml).

 

Fig 4. Calibration graph of Ibuprofen for  Fixed time method:

 

Fig 5. Calibration graph of Nimisulide for  Fixed time method

 

Fixed Concentration method:

A preselected value of the absorbance was fixed and the time was fixed and the time was measured for different Drugs concentrations (Table 2).The time versus the initial Concentration of Anti inflammatory drugs was plotted, which could be used as a calibration graph. The following equation was obtained:

T= -8E.06 x + 0.000 [Ibuprofen] (r=0.988)

T= -3E.06 x + 0.000 [Ibuprofen] (r=0.990)

 

The range of the drug concentrations giving the most acceptable calibration graph with the above was very limited which could be a disadvantage.

 

 

Table 1 .Various Kinetic methods for the determination of Ibuprofen

 

[IB]x 103/

mol dm-3

Rate constant method

K obs x 103 / S-1

Fixed concentration method

(Abs = 0-15)

Fixed time method

(t = 120 s) Abs.

610 nm

526 nm

610 nm

526 nm

610 nm

526 nm

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

6.82

7.09

7.25

7.43

7.61

7.84

7.96

-

-

-

4.1

3.4

2.7

2.1

1.5

1.1

-

-

-

-

90

70

50

30

20

-

-

-

-

-

210

180

140

110

90

-

-

-

-

-

3.2

3.4

3.5

3.6

3.7

3.8

3.9

4.0

4.1

4.2

6.5

6.2

5.9

5.6

5.4

5.1

4.8

4.7

4.5

4.1

a. Experimental and calculated.

 

 


Table 2. Various Kinetic methods for the determination of Nimesulide

[NIME]x 103/

mol dm-3

Rate constant method

K obs x 103 / S-1

Fixed concentration method

(Abs = 0-15)

Fixed time method

(t = 120 s) Abs.

610 nm

526 nm

610 nm

526 nm

610 nm

526 nm

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

4.48

5.39

6.28

7.40

8.32

9.42

9.89

-

-

-

9.1

7.9

6.3

5.1

4.2

3.3

2.2

-

-

-

300

250

210

190

130

90

-

-

-

-

210

180

140

110

90

-

-

-

-

-

3.24

3.40

3.55

3.63

3.82

3.99

4.13

4.22

4.30

4.43

5.6

5.3

5.1

4.8

4.6

4.4

4.2

4.0

3.9

3.7

a. Experimental and calculated

 

Table 3: Analytical parameters of Ibuprofen drug with Alkaline KMno4.

Drug/ Methods

Wave

length

Linear

Range(µg/ml)

Intercept

Correlation coefficient(R2)

LOD

LOQ

Sandell’s sensitivity

Ibuprofen

A. Rate

constant

method

610

 

526

20.6-123.7

 

20.6-123.7

0.00074

 

0.00052

0.9970

 

0.9963

0.1429

 

0.0485

0.4330

 

0.1470

0.0037624

 

0.0077838

B. Fixed

time

method

610

 

526

20.6-206

 

20.6-206

0.3178

 

0.6193

0.9915

 

0.9967

0.012

 

0.021

0.0041

 

0.0032

0.0000057

 

0.0000058

 

Table4: Analytical parameters of Nimesulide drug with Alkaline KMno4.

Drug/ Methods

Wave

length

Linear

Range(µg/ml)

Intercept

Correlation coefficient(R)

LOD

LOQ

Sandell’s sensitivity

Nimesulide

A.  Rate constant

method

610

 

526

30.6- 215.8

 

30.6- 215.8

0.0044

 

0.0018

0.9973

 

0.9953

0.0287

 

0.0236

0.0868

 

0.0714

0.0044402

 

0.0038612

B.  Fixed

Time method

610

 

526

30.8-306

 

30.8-306

0.3139

 

0.671

0.9950

 

0.9955

0.00025

 

0.00013

0.00063

 

0.00030

0.0000095

 

0.0000067

 


Initial Rate method:

In this method, graphs of the rate (at the beginning of the reaction) versus the drug concentration were not easy to obtain because the reaction was fast. Thus, the tangents to the curves at zero time were not easy to draw. This method was therefore abandoned.

 

The best correlation coefficient was obtained for the Fixed time method, and the value of the slope was also high. Even though the range was limited compared to the rate-constant method, the Fixed-time method was found to be more applicable.

 

Statistical analysis of the results in comparison with the official method:

The performance of the proposed method was judged by calculating the student t-test and variance ratio F-test. At the 95% confidence level, the calculated t- test and F-values do not exceed the theoretical values (Table 5 and 6), indicating that there is no significant difference between the proposed method and the official method. From an analytical point of view, it is concluded that the described procedure allows for the determination of Anti Inflammatory drugs in pure and pharmaceutical dosage forms. Unlike the spectrofluorometer, as well as gas chromatographic and HPLC procedures, the instrument is simple and inexpensive. Its importance lies in the chemical reaction upon which the procedure is based, rather than sophistication of the instrument. This aspect of the kinetic method of determination is of major interest in analytical pharmacy, since it offers a distinct possibility for the assay of a particular component in complex dosage formulations.

 

3.4: Validation of the proposed method:

Concentration range is established by confirming that the analytical kinetic procedures provides a suitable degree of precision, accuracy and linearity when applied to the sample containing the amount of analyte within or at the extreme of the specified of the range of the analytical procedure. In this work,, concentrations ranging from 20.6 – 123.7 µg/ml and 30.6 – 215.8 µg/ml were studied for the investigated drugs in the Rate constant method and concentration ranging from 20.6-206 µg/ml and 30.6 - 308 µg/ml were studied for the investigated drugs in the constant time method (at preselected fixed time for 240 secs).The whole sets of experiments were carried out through this range to ensure the validation of the proposed procedure. Linear calibration graphs were obtained for all the studied drugs by plotting the logarithm of rate constant method of the reaction versus Absorbance of molar concentration of analyte in the sample within the specific range.

Table 5: Analysis of Ibuprofen in pharmaceutical formulations

(Found valuesa ± SD% and comparison with the official method).

Drug

 

Labeled

 

Found (X ± SD)

Proposed method    Reference method

Tablets:

Brufen

 

Ibugin

 

Ibupal

 

Ibrumac

 

Capsules:

Ibubid

 

Ibugesic

 

200 mg

 

200 mg

 

400 mg

 

400mg

 

 

300 mg

 

300mg

 

 

198 ± 0.71

t = 0.32, F = 1.39

198 ± 0.65

t = 0.42, F = 1.09

398 ± 0.74

t = 0.82, F = 0.39

399 ± 2.5 mg

t = 0.25, F = 1.99

 

298 ± 2.3 mg

t = 0.22, F = 2.01

299 ± 1.9 mg

t = 0.25, F = 1.99

t = 0.5, F = 2.19

 

200.98 ± 0.43

 

199.98 ± 0.13

 

399.98 ± 0.23

 

400.2 ±  1.27 mg

 

 

300.5 ± 0.97 mg

 

300.2 ±  1.01 mg

 

 

Table 6: Analysis of Nimesulide in pharmaceutical formulations

(Found valuesa ± SD% and comparison with the official method).

Drug

 

Labeled

 

Found (X ± SD )

Proposed method    Reference  method

Tablets

 

 

 

Antipen

 

Nicip

 

Nise

 

Nimulase

 

100 mg

 

50 mg

 

100 mg

 

10 mg

 

97 ± 0.78

t = 0.12, F = 0.59

49 ± 0.84

t = 0.92, F = 1.49

98 ± 0.34

t = 1.2, F = 0.93

89.21 ± 1.12

t = 0.2, F = 1.9

100.2 ± 0.44 mg

 

52.2 ± 0.32 mg

 

99.21 ±  0.56 mg

 

90.34 ± 0.21mg

 

Syrups

 

 

 

Cunim

 

Relisulide

 

60 ml

 

60 ml

59.87±1.23

t= 1.5, F=0.78

58.98±0.99

t=1.34, F=0,97

60.32±0.33 mg

 

60.59± 0.47 mg

 

Precision was checked at three concentration levels. Eight replicate measurements were recorded at each concentration level. The calculated relative standard deviation were all below 2.2% indicating excellent precision of the proposed procedures at both level of repeatability and intermediate precision.

 

Limit of detection (LOD): was calculated based on standard deviation of response and the slope of calibration curve. The limit of detection was expressed as,

                       LOD= 3σ  

                                  S

Where σ is the standard deviation of intercept s is the slope of calibration curve. The results were summarized in (Tables 3 and 4) indicating good sensitivity of the proposed method .According to USP XXV guidelines, the calculated LOD values should be further validated by laboratory experiments. In our work, good results were obtained where the calculated by LOD equations were actually detected in these experiments.

 

Limit of Quantification (LOQ): was calculated based on standard deviation of intercept and slope of calibration curve. In this method, the limit of quantization is expressed as

LOQ= 10σ  

                S

The results were summarized in (Table 3 and 4) indicating the good sensitivity of the proposed method. According to USP XXV guidelines, the calculated LOQ values should be further validated by laboratory experiments. In our work, good results were obtained where the calculated by LOQ equations were actually quantities in these experiments.

 

3.5: Application to pharmaceutical Dosage forms:

The Fixed time method and rate constant methods of the proposed kinetic spectrophotometric method for the investigated drugs have been tested on commercial pharmaceutical dosage forms. The concentration of investigated drugs was computed from its responding regression equations. The results of proposed method (Fixed time and rate constant methods) were statistically compared with those of reported methods, in respect to accuracy and precision. The obtained mean recovery values were recorded in (Table 5 and 6), which ensures that there is no interference of other additives present in the studied formulations.

 

In the t- and F- tests, no significant differences were found between the calculated and theoretical values of both the proposed and the reported methods at 95% confidence level. This indicates good precision and accuracy in the analysis of investigated Antifungal drugs in dosage forms.

 

4. CONCLUSION:

The Fixed time and rate constant methods can be easily applied for determination of investigated anti inflammatory drugs in pure and dosage forms that do not require elaborate treatment and tedious extraction of chromophore produced. The proposed methods (Fixed time and rate constant method) are sensitive enough to enable determination of lower amounts of drug; these advantages encourage the application of proposed method in routine quality control of investigated analgesic drugs in industrial laboratories. Finally our methods provides advantage of improving selectivity, avoiding interference of colored and/ or are turbidity background of samples because it measures the increase in absorbance with time against blank treated similarly.

 

5. ACKNOWLEDGEMENT:

Corresponding author thanks Principal and Management, GMIT Davangere for their support in carrying out this work. Also author would like to thank Mr. Girish Bolakatte, Lecturer, Dept. of Chemistry, Bapuji Pharma College, Davangere for his assistance in carrying out this work.

 

6. REFERENCES:

1.       The merck index, 12th ed., merck and Co., Whitehouse Station, NJ. 1996: 1125.

2.       Alvarez-Lueje A, Vasquez P, Nunez-Vergara L J,and squella J A. electroanalysis.1997:9:1209-1213.

3.       Alvarez-Lueje A, Vasquez P, Nunez-Vergara L J, and squella J A. anal. Lett. 1998: 31: 1173-1184.

4.       Laksmi C S R, reddy M N, and Naidu P Y. Indian drugs. 1998:35:519-520.

5.       Navalgund S G, Prabhu P S, Sahasrabudhe P S, D H Khanolka, and R T sane. Indian drugs.2000: 37:209-210.

6.       Robert Bello F P, and Evasherman S E, Rev. Bras. Farm. 1995:30:76-80.

7.       Rajputh S J, and randive G, The East pharm. 1997:40:396-398.

8.       Choudri K P R, Rao G D, and Babu I S, Indian drugs. 1997:34:396-398.

9.       Choudri K P R, Rani A R, and Latha L S, The East Pharm, 1998: 41:163-165.

10.     Reddy M N, Reddy K S, Sankar D G, and Sridhar K, The East Pharm. 1998:41:119-121.

11.     Lakshmi C S R, and Reddy M N, Mikrochem.Acta. 1999:132:1-6.

12.     Girish Kumar K, Chowadary K P R, and Rao G D, Indian Drugs. 1999:36:185-186.

13.     Girish Kumar K, Chowadary K P R, and Rao G D, Indian J Pharm Sci. 1999:61:86-89.

14.     Reddy M N, Murthy T K, and Sankar D G, The East Pharm. 2001:44:121-122.

15.     M N Reddy, T K Murthy D G Sankar. Asian J.Chem. 2001:13:915-918.

16.     Nagaraja P, Srinivasamurthy K C, and Yathirajan H S, talanta. 1996:43:1075-1080.

17.     Nagaraja P, Srinivasamurthy K C, H S Yathirajan, and  Mohan B M, Indian J Pharm. Sci. 1998:60:99-101.

18.     Nagaraja P, Sunitha K R, and Silwadi M F, J. Pharm. Biomed. Anal. 2000:23:617-622.

19.     Nagaraja P, Vantha R A, and Sunitha K R, J. Pharm. Biomed. Anal. 2001:25:417-424.

20.     Kanoute G, Boucly P, Guerent Nivand E, and Guernet M. Ann. Pharm. Fr. Oct. 1985:43:265-272.

21.     Kanoute G, Nivand E, Paulet B, and Boucly P. Talanta. 1994:31:144-146.

22.     Lu J, Wang M, Wu A. and Qiu X, Shanga Yika Daxue Xuebao. 1992:17:275-277.

23.     Mahrous M S, Abdel Khalek M M, and Abdel Hamid M E, J. Assoc. Off. Anal. Chem. 1985:68:535-539.

24.     El-Din M K S, Abuirjeie M A, and Abdel-Hay M H, Anal. Lett. 1991:24:2187-2706.

25.     Matsuda R, Takeda Y, Ishibashi M, Uchiyama M, Suzuki M, and  Takitani S, Bunseki Kaguku. 1986:35:151-156.

26.     Abdel-Hay M H, Korany M A, Bedair M M,  and Gazy A A, Anal. Lett. 1990:23:281-294.

27.     Husain S, Srk A. Murthy, and Rao A R, Indian Drugs. 1989:26:185-189.

28.     El Ragehy N A, Bulletin of fac. of pharmacy. 1991:29:1-4.

29.     Ge J, and  He Y Zhonggue Yaoke Daxue Xuebao. 1987:18:88-95.

30.     Rao G R, Avadhanulu A B, and Pantulu A R R, East Pharm. 1991:34:119-121.

31.     Pant S K, and G L Jain, Indian Drugs. 1991:28:216-265.

32.     Rustom A M, J.Chromato. Ghr. Sci. 1991:29:16-20.

33.     Goodall D M, Riley M J, Wu Z, and Wilson I D, Anal. Proc. 1992:29:253-255.

34.     Haikla V E, Heimonen L K, and Vuorela H J, J. Pharm. Sci. 1991:80:456-458.

 

 

 

 

Received on 28.12.2010        Modified on 18.01.2011

Accepted on 08.02.2011        © AJRC All right reserved

Asian J. Research Chem. 4(4): April, 2011; Page 659-665