Spectrophotometric Determination of Diethylcarbamazine and Centperazine in Urine S. K. Baveja and K. V. Ranga Rao Department of Pharmaceutical Sciences, Panjab University, Chandigarh-160 014, India Keywords : Microfilaracidal drugs ; diethylcarbamazine ; centperazine ; spectrophotometry ; picrolonic acid Diethylcarbamazine (l-diethylcarbamoyl-4-methylpiperazine, Banocide, Hetrazan) has been widely used in the control and treatment of human filariasis for the last 30 years. Cent- perazine (3-ethyl-8-methyl-l,3,8-triazabicyclo [4.4.0]decan-2-one), an analogue of diethyl- carbamazine with greatly reduced conformational mobility, was found to be more potent1 (four times orally and six times intraperitoneally) against microfilariae than diethyl- carbamazine. Further, the therapeutic index of centperazine is much higher than that of diethylcarbamazine and it possesses antihistaminic and anti-inflammatory properties that are desirable in the treatment of frlariasis.At present centperazine is undergoing extensive clinical trials. To help in understanding the mode of action of such drugs a sensitive, accurate and simple method of determination is essential. The methods currently in use2--5 for the assay of diethylcarbamazine depend on the formation of coloured complexes with sulpho- naphthalein indicators that can be extracted into an organic solvent. More recently two other methods, the first based on the reaction with picric acids and the second the phenol reagent (Folin - Ciocalteu reagent) method,' ,have been reported for the quantitative deter- mination of diethylcarbamazine.Picro- lonic acid [2,4-dihydro-5-methyl-4-nitro-2-(4-nitrophenyl)-3H-pyrazol-3-one] was found to form complexes with diethylcarbamazine and centperazine, which could then be used to determine them quantitatively at 350 and 354 nm, respectively. Recovery studies revealed that this method could also be used to determine the drugs in urine. The experimental procedure and results are reported in this paper. All of the methods listed are of low sensitivity. Experimental Apparatus vortex mixer was used for shaking. A Hitachi Perkin-Elmer, Model 200, spectrophotometer was used. A Labaids miniorb Reagents hydroxide solution and 0.1 M acetate buffer solution (pH 6). Bonner's reagent. Mix equal volumes of 0.2 M sodium chloride solution, 10 N potassium 726June, 1981 SHORT PAPERS 727 Acetic acid, 2 N.Picrolonic acid reagent. Prepare freshly by diluting 10ml of a saturated solution of picrolonic acid to 20 ml with 0.1 M acetate buffer solution (pH 4.6). Wash the solution with four 20-ml portions of 1,2-dichloroethane. Standard solutions of diethylcarbamazine citrate and centperazine. Prepare aqueous solutions containing 10-240 pg ml-l of each compound (multiples of 10 pg ml-l) by diluting a stock solution of 400 pg ml-l with water. Procedure Aliquots (1 ml) of each of the series of dilute standard solutions (containing 10-240 pg ml-1 of drug) were pipetted into separate glass-stoppered tubes. To each of the tubes was then added 1 ml of Bonner’s reagent and the solutions were mixed gently.1,2-Dichloroethane (5d) was then added and the contents were shaken vigorously for 3-5min using the vibrator and centrifuged at 3000revmin-’ for 10min. The upper aqueous layer was removed by suction and discarded, and the lower organic layer was decanted into another glass-stoppered tube containing 0.1 ml of 2 N acetic acid. The solution was thoroughly mixed so that any residual alkali present in the organic layer was neutralised. Picrolonic acid reagent (1 ml) was then added and the mixture shaken for 3 min and again centrifuged. The aqueous phase was aspirated and discarded, and the absorbance of the organic phase was measured at 350 nm for diethylcarbamazine citrate and 354 nm for centperazine. The blank was prepared by the same procedure except that the drug solution was replaced with distilled water, Calibration graphs were drawn from the results obtained. Recovery of added drug from urine An aliquot (1 ml) of each of the dilute standard solutions was transferred into a glass- stoppered tube, mixed with 1 ml of urine and allowed to stand for 2 h for reaction to occur.The procedure described previously was then followed, commencing with addition of Bonner’s reagent to each tube. Results and Discussion Absorbance maxima of the complexes of diethylcarbamazine and centperazine with picrolonic acid (Fig. 1) were found to be at 350 and 354 nm, respectively. There was a good linear relationship between the mass of drug taken and the absorbance in the range 0-240 pg for diethylcarbamazine citrate and 0-180 pg for centperazine.The complexes were found to be stable for at least 4 h. The method is sensitive Calibration graphs are shown in Fig. 2. 1.4 - 1.2 - 0, (D 2 1.0 - 0.8 - 9 a 0.6 - 0.4 - 0.2 - 220 240 260 280 300 320 340 360 W avelengthh m Fig. 1. Absorption spectra of A, complex of diethylcarbamazine and picrolonic acid in 1,2-dichloroethane and B, complex of centperazine and picrolonic acid in 1,2-dichloro- ethane. 2.8 2.4 8 2.0 e 2 1.2 [I (D 1.6 2 0.8 0.4 0 Amount of druglyg Fig. 2. Calibration graphs of A, di- ethylcarbamazine (1 pg of diethylcar- bamazine = 1.97 pg of diethylcarbamazine citrate) and B, of centperazine.728 SHORT PAPERS Analyst, Vol. 106 to as little as 1 pg ml-l of the drugs. The A izi values for the complexes of diethylcarbamazine citrate and centperazine by this method were found to be 1190 and 695, respectively (com- pared to the organic phase).This value for the former is much higher than that obtained by earlier methods,2-7 indicating the much greater degree of sensitivity attained. Table I shows the recoveries obtained when 10-180-pg amounts of diethylcarbamazine citrate and centperazine were added to 1 ml of urine as described above. The results obtained were analysed using the Student’s t-test. The t values calculated are less than the tabulated values for to.01,6, and it is evident that the mean assay results do not differ significantly from the true contents. Diethylcarbamazine Centperazi ne Picrolonic acid TABLE I RECOVERY OF DIETHYLCARBAMAZINE CITRATE AND CENTPERAZINE (IN MICROGRAMS) FROM URINE BY THE PROPOSED METHOD Experiments were performed a t room temperature (35 f 1 “C).Amount of drug added/pg Diethylcarbamazine citrate Cen tperazine * I c ‘5 10 20 40 80 120 160 186 5 10 20 40 80 120 160 120 5.38 10.75 18.64 43.13 81.20 119.58 163.03 183.40 5.20 9.34 20.79 39.42 79.26 118.57 157.93 180.77 5.46 9.62 20.07 38.35 81.11 113.72 154.01 175.90 4.71 10.48 20.43 39.71 80.73 123.57 156.45 176.90 4.62 10.45 21.35 37.53 75.63 120.41 159.75 174.27 4.55 10.40 19.20 42.50 77.06 117.71 153.50 171.50 4.77 10.83 18.79 41.48 81.37 117.90 152.38 174.27 4.47 9.18 20.57 41.29 81.46 120.71 160.88 172.27 4.77 9.47 21.28 39.34 75.80 119.58 158.93 182.75 4.72 10.40 19.85 39.14 75.59 119.28 153.50 172.27 5.15 9.85 18.41 41.97 77.25 114.56 150.83 175.93 4.80 9.67 20.43 40.86 80.00 117.85 156.45 178.45 Mean .. . 5.03 10.16 19.76 40.30 78.73 117.63 156.49 177.76 4.74 9.91 20.21 40.49 79.02 119.62 156.45 175.36 Standard deviation :. 0.35 0.59 1.34 2.22 2.80 2.83 4.78 4.19 0.26 0.58 0.59 1.30 2.26 4.26 2.80 3.88 t + .. .. .. 0.21 0.66 0.44 0.33 1.11 2.05 1.79 1.31 2.55 0.35 0.87 0.92 1.06 0.22 3.11 2.93 * t values were calculated for the whole of the data and these values for diethylcarbamazine citrate and centperazine were 0.124 and 0.127, respectively. These are less than the tabulated values for t,.,,, ,*. We are grateful to Dr. Nitya Anand, Director of the Central Drug Research Institute, Provision of a sample of diethyl- Lucknow, India, for the generous supply of centperazine. carbamazine citrate by Unichem Laboratories Ltd., Bombay, is acknowledged. References 1. Saxena, R., Iyer, R. N., Anand, N., Chatterjee, R. K., and Sen, A. B., J . Pharm. Pharmacol., 1970, 22. 306. I ~ - ~ - 2. 3. 4. 5. 6. 7. Lubran, M., Br. J . Pharmacol., 1950, 5, 210. Basu, K., and Dutta, B. N., Indian J . Pharm., 1961, 23, 326. Vadodaria, D. J., Vora, M. N., and Mukherji, S. P., Indian J . Pharm., 1968, 30, 41. Rao, K. N., and Subramanyam, D., Indian J . Med. Res., 1970, 58, 746. Ramachandran, M., Indian J . Med. Res., 1973, 61, 864. Chandrasekaran, B., Patil, S. K. B., and Harinath, B. C . , Indian J . Med. Res., 1978, 67, 106. Received December 31st, 1979 Accepted December 2nd, 1980