New Analytical Methods for the Assay of Flupentixol in Bulk Samples and Pharmaceutical Formulations
Saumindra P. Das1, Pradyusa Samantray2, Baidhar Sahoo3 and Swoyam P. Rout1*
1Department of Chemistry, Utkal University, Vani Vihar, Bhubaneswar 751004
2Department of Chemistry, Indian Institute of Technology, Bhubaneswar 751013
3Department of Chemistry, Ravenshaw University, Cuttack 753003
*Corresponding Author E-mail: swoyamrout@gmail.com
ABSTRACT:
Novel visible spectrophotometric methods having better sensitivity, selectivity, precision and accuracy have been developed for the determination of Flupentixol (FLU), an antipsychotic drug by exploring its various analytically useful functional groups.
KEYWORDS: FLU, Spectrophotometric methods.
Flupentixol (FLU) is chemically known as 4-{3-[2-(Trifluromethyl)-9H-thioxanthen-9-ylidene] propyl} -1 piperazineethanol. (C23 H25F3N2O S) and is used for the treatment of hallucinations, delusions and other thought disorders associated with schizophrenia.
Very few physico -chemical methods have been reported in the literature for the determination of FLU in Pharmaceutical formulations. The methods reported mainly include LCMS1, GC-MS2, Fluorimetry3, HPLC4, HPTLC5 and GC6.
Literature survey reveals that the analytically important functional groups of FLU have not been properly exploited for designing suitable spectrophotometric methods for its determination and thus there are very few reports, reported so far, utilizing visible spectrophotometric technique. Hence the authors have made an attempt to develop visible spectroscopic determination of FLU as the said method is simple, sensitive, and accurate and of low cost.
Several methods have been developed by the authors based on the reactivity of FLU with different reagents to produce coloured species of reasonable stability thus providing possibility for spectrophotometric determination of FLU in bulk samples and pharmaceutical formulations.
The authors have also developed a simple and sensitive UV method (using methanol as solvent) as a reference method for comparing accuracy of the results obtained by the proposed methods.
Out of several methods developed by the authors, two methods based on chemical reaction of FLU with reagents such as Chloramine-T (CAT)/ Gallocyanine (GC ) (Method-I) and Wool Fast Blue (WFB BL), (Method II) have shown best results in terms of sensitivity. These two proposed methods also have high λ max values which are of a decisive advantage since the interference from the associated contaminants shall be far less at higher wavelength, the results of which are reported here.
MATERIALS AND METHODS:
Systronics 166 digital spectrophotometer with 1 cm matched quartz cells were used for the spectral and absorbance measurement. Digital pH meter Systronics 361 was used for pH measurement.
Preparation of standard solution:
The stock solution (mg/ml) of Flupentixol as hydrochloride was prepared by dissolving 100 mg of Flupentixol in 100 ml of distilled water. A portion of the stock solution was diluted stepwise with the same solvent to obtain the working standard of FLU solution of, concentration 50 µg/ml (for Method I ) and 40 µg/ ml(for Method II ).
Preparation of Reagent:
All the chemicals and reagents prepared were of analytical grade and double distilled water was used to prepare the solutions.
For method I
|
CAT solution ( Loba;0.02 % ,7.10 x 10-4M ) |
Prepared by dissolving 20 mg of Chloramine-T (CAT) in 100ml of distilled water and standardized iodometrically. |
|
GC solution ( Chroma,0.01% ,2.269 x 10-4M ) |
Prepared by dissolving 10mg of Gallocyanine (GC) in 100 ml of distilled water. |
For method II
|
WBF BL solution (Flukas,0.2% ,3.26 x 10-3M) |
Prepared by dissolving 200mg of Wool Fast Blue BL (WBFBL) in 100 ml of distilled water. |
|
Buffer solution of pH =1.5 |
Prepared by mixing 289 ml of glycine solution (37.52 g of glycine and 29.24 g of NaCl were dissolved in 500 ml distilled water) with 711 ml of 0.1 M HCl and pH of the solution was adjusted to 1.5. |
Procedure:
For Bulk Sample:
Method I:
Aliquots of FLU solution (1.0-3.0 ml, 50 µg /ml) were transferred into a series of 25 ml graduated tubes. Then 1.25 ml of 5 M HCl followed by 2.0 ml of 0.02% CAT were added to each tube and solution was diluted to 20 ml with distilled water. After 10 minutes, 4 ml of GC solution (0.01%) was added and the absorbances were measured after 15 minutes at 540 nm against similar reagent blank. The decrease in absorbance corresponding to consumed CAT(in turn to drug quantity) was obtained by subtracting the absorbance of the blank solution from that of the test solution. The calibration graph was drawn by plotting the decrease in the absorbance of the dye (GC), against the amount of drug and the amount of drug in any sample was derived from its calibration graph (i.e Beer’s law plot of FLU with CAT/GC system, absorbances vs. concentration (µg/ml).
Method II:
1 to 3 ml of buffer solution (pH 1.5) and 2 ml of WFB BL dye solution were added to a series of 125 ml separating funnels containing aliquots of standard FLU solution (0.5-2.5 ml 40 µg/ml) .The total volume of aqueous phase in each separating funnel was adjusted to 15 ml with distilled water and 10 ml of chloroform(CHCl3) was added. The contents were shaken for 2 minutes and the two phases were allowed to separate. The absorbance of the separated organic layer was measured at λmax =580 nm against a similar reagent blank. The amount of FLU was computed from the approximate calibration curve [Beer’s Law plot of FLU with WFB BL system-Absorption vs. concentration (µg/ml)].
For Pharmaceutical Formulations:
The tablet powder equivalent to 100 mg of FLU was taken and triturated with (3x 25 ml) portions of methanol. The combined methanol extract was made upto 100 ml with the same solvent to get the stock solution. (mg/ ml).
From a portion (i.e 20 ml of methanol extract), methanol was gently evaporated. The residue was dissolved in distilled water and then the volume was brought to 50 ml with the same solvent to get 400 µg/ml of sample. It was further diluted stepwise with the same solvent to obtain 50 µg/ml sample (for Method I) and 40 µg/ml (for Method II).
Then the procedure given under bulk samples were followed for the assay of FLU in formulation.
Reference Method:
Accurately weighed portion of the powered tablets equivalent to 100 mg of drugs was dissolved in 20 ml of methanol, shaken well and filtered. The filtrate was diluted to 100 ml with methanol to get 1 mg/ml of drug solution in formulations. 2 ml of this solution was further diluted to 200ml to get 10 µg/ml solutions. The absorbance of the solution was determined at λmax 290 nm. The quantity of drug was computed from the Beer’s law plot (Absorbance vs. wavelength) of the standard FLU in methanol.
RESULTS AND DISCUSSION:
In order to ascertain the optimum wave length of maximum absorption (λmax) of the coloured species formed in the proposed two methods, 5 µg /ml of FLU in Method 1 and 8 µg /ml of FLU in Method II in final solution were taken and respective colours were developed separately. The absorption spectra were scanned on a spectrophotometer in the wave length range of 360-900 nm against the similar reagent blank. The reagent blank absorption spectrum of each method was also recorded against distilled water. The absorption curves of coloured species formed in each method showed characteristics absorption maxima where as blank in each method showed low absorption in this region.
Method I involves a two steps reaction, namely reaction of FLU with an excess amount of oxidant(CAT) and estimation of unreacted oxidant using a known excess of dye GC. The excess dye remaining was then measured with a spectrophotometer at 540 nm. The effect of oxidant concentration and acidity at different intervals in first step and dye concentration in second step, waiting period in each step with respect to maximum sensitivity, minimum blank, adherence to Beer’s Law, reproducibility and stability of coloured species formed after final dilution were studied through control experiments in order to establish optimum conditions for colour development. In Method II, the optimum condition for the colour development was established basing on the study of effects of various parameters such as type of buffer, concentration of dye WFB BL, choice of organic solvent, ratio of organic phase to aqueous phase, shaking time and temperature, intensity and stability of the coloured species in organic phase by measuring the absorbances at λ max = 580 nm.
Table 1: Optical and regression characteristics, precision and accuracy of the proposed two methods.
|
Sl. no |
Parameters |
Method 1 (using CAT/ GC as reagent) |
Method II ( using WFBBL as reagent) |
|
1 |
λmax(nm) |
540 |
580 |
|
2 |
Beer’s law limits(µg / ml) |
2-6 |
2-10 |
|
3 |
Molar absorptivity (mol-1cm-1) |
5.544 x 104 |
3.207 x 104 |
|
4 |
Sandell’s sensitivity (µg / cm2/0.001 absorbance units) |
0.009 |
0.01578 |
|
5 |
Optimum photometric range(µg/ml) |
2.5-5.4 |
3.2-8.4 |
|
6 |
Regression equation(y=a+bc) i) Slope(b) ii) Standard deviation on slope(Sb) iii) Intercept(a) iv) Standard deviation on intercept(Sa ) v) Standard error of Estimation (Se ) |
0.10930 0.00091 -0.00020 0.00388 0.00289 |
0.06330 0.00051 -0.00160 0.00338 0.00322 |
|
7 |
Correlation coefficient( r) |
0.9998 |
0.9999 |
|
8 |
Relative standard deviation * |
0.2739 |
0.2459 |
|
9 |
% Range of error(confidence limits) i)0.05 level ii)0.01 level |
0.229 0.339 |
0.206 0.304 |
|
10 |
% error in bulk sample ** |
0.138 |
0.025 |
* Average of six determinations. ** Average of three determinations.
In order to test, whether the colored species formed in the above two methods adhere to Beer’s law, the absorbance at appropriate wavelength of a set of solutions containing different amounts of FLU and specified amount of reagents (as described under experimental procedure of the methods) were recorded against appropriate reagent blanks. Beer’s law limits, molar absorptivity, Sandell’s sensitivity and optimum photometric range for FLU with each of the mentioned reagents were calculated.
Least square regression analysis was carried out for the slope, intercept and correlation coefficient. The precision of the aforesaid methods were ascertained separately from the absorbance values obtained by actual determination of six replicates of a fixed amount of FLU i.e 5 µg / ml (Method I) and 8 µg / ml (Method II)in final solution. The percent relative standard deviation and percent range of error (at 0.05 and 0.01 confidence limits) were calculated for the aforesaid two methods.
To determine the accuracy of the aforesaid methods, different amount of bulk samples of FLU within the Beer’s law limits were taken and analyzed by the proposed methods. The result (i.e percentage error) was recorded. All the above results are shown in Table 1.
The effect of wide range of inactive ingredients usually present in the formulations for the assay of FLU under optimum conditions were investigated .None of them were found to interfere in the methods developed even if present in excess amount than anticipated in pharmaceutical formulations.
CONCLUSION:
Looking at the determination capability, extraordinary detection sensitivity, rapidity, simplicity, low cost and accuracy, the two proposed spectrophotometric methods can be recommended for such analysis.
REFERENCES:
1. McClean.S., O’Kane.E.J., Smyth.W.F., J.Chromatogr., B.Biomed. Sci Appl. 2000 : 740(2) : 141-157.
2. Maurev. Hans., Pfleger. Karl., J.Chromatogr. 1984 : 306 : 125-45.
3. Shehata .I.A., EL-Ashry.S.N., EL-Sherbeny.M.A., EL-Sherbeny. D.T., Belal.F., J.Pharm. Biomed. Anal. 2000 : 22(5) : 729-737.
4. Angelo.Helle.Riis., Herrstedt.Joesn., Joergensen. Morten., J.Chromatogr.1989: 496(2): 472-7
5. Po.A.Li Wan., Irwin.W.J., HRC CC.J.High Resolut. Chromatogr. Commun. 1979: 2(10): 623-7.
6. Blant-Gorgia. A.E., Balan .L.P., Gent.C , Eisele. R., Ther. Drug Monit. 1985 : 7(2 ) : 229-35.
Received on 29.07.2010 Modified on 08.08.2010
Accepted on 15.08.2010 © AJRC All right reserved
Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 1083-1085