482 Autalyst, August, 1974, Vol. 99, $9. 482-486 Colorimetric Determination of Piperazine in Pharmaceutical Formulations BY YEHIA M. DESSOUKY AND SAAD A. ISMAIEL (Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Caivo, Egypt) (Research Department, SociLtte' Misr pour l'lndustrie Pharmaceutique, 92 El Mataria Street, Post El Zeitoun, Cairo, Egypt) Piperazine can be satisfactorily determined in pharmaceutical prepara- tions or formulations such as effervescent granules and elixirs containing hexamine, colchicine, atropine sulphate, sodium benzoate, lithium benzoate, lithium citrate, sodium citrate, sodium hydrogen carbonate, tartaric acid, citric acid, lactose, sucrose and Tinct. ammi visnaga. The diluted sample solution is treated with a 0.6 per cent.aqueous I ,2-naphthoquinone-4-sulphon- ate solution in the presence of acetate - citrate buffer a t pI3: 7-5. The tempera- ture of the reaction should be between 10 and 15 "C and the colour produced is measured a t 490nm. PIPERAZINE, a pyrazine derivative, is one of the most potent drugs used as an anthelmintic for the treatment of threadworms and roundworms in both children and adults, the worms usually being voided from the host while the drug is still active. Piperazine in effervescent granules cannot be determined by using either the non-aqueous titration method of the United States Pharmacopeial or the gravimetric method of the British Pharmacopoeia.2 Further, the colorimetric methods of Pankrat~,~ who used the reineckate salt, and Perlm~tter,~ who used acidified 9-benzoquinone, could not be applied to this preparation.This inapplicability is because of the presence and interference of hexamine, citric and tartaric acids, sodium citrate, sodium hydrogen carbonate and other interfering ingredients that yield either higher or lower results. The titrimetric f ormol method suggested by Simionovici, Rsianu and Cuculescu5 also cannot be used for an accurate determination of piperazine in coloured pharmaceutical preparations as the colour masks the end-point, giving high results. Gravimetric methods, such as those described by Maynard6 for the diacetyl derivative and by Chemerisskaya' for the dichromate derivative were tried, but accurate results were not obtained as the methods are not sensitive. They are time consuming, however. In the present investigation, a colorimetric method for the determination of piperazine in some pharmaceutical preparations is proposed that involves the use of the red colour formed on treating piperazine with 1,2-naphthoquinone-4-sulphonate in the presence of acetate - citrate buffer at pH 7.5.The proposed method is free from the drawbacks of the above methods.l-' REAGENTS- The pure piperazine used was of pharmaceutical grade and all of the chemicals and reagents were either of analytical-reagent or pharmaceutical grade. 1,2-Naphthoquinone-4-suZ~honate reagent-Dissolve 0.3 g of 1,2-naphthoquinone-4-~~1- phonate sodium salt in 50ml of water. Acetate - citrate bufler, pH 7.5-Dissolve 5.0 g of sodium acetate and 1.0 g of sodium citrate in about 50 ml of water and adjust the pH of the solution to 7.5 with a few drops of 33 per cent.acetic acid, then dilute to a volume of 100 ml with water and filter if necessary. Standard solution-Weigh accurately 100 rng of piperazine citrate (or hexahydrate, depending on the formulation) and dissolve it in sufficient water to produce 100 ml. Dilute 3 ml of this solution to 100 ml with water. Samples-The composition of each of the samples tested is given in Table I. @ SAC and the authors. EXPERIMENTAL This reagent must be freshly prepared.DESSOUKY AND ISMAIEL TABLE I FORMULATION OF ANALYSED SAMPLES Sample 483 Constituent Piperazine hexahydratelg . . Piperazine citratelg . . .. Hexamine/g . . . . .. Colchicine/g . . .. .. Atropine sulphatelg . . .. Sodium benzoatelg .. .. Lithium benzoate/g . . .. Lithium citrate/g . . . . .. Sodium citratelg . . .. .. Tartaric acid/g . . .. . . Citric acid/g . . .. .. Lactose/g . . .. .. .. Sucroselg . . .. .. .. Tinct. ammi visnaga/ml . . Sodium hydrogen carbonatelg . . .. Coloured flavoured syruplml . . Flavoured, coloured effervescent mixturelg . . .. .. Effervescent mixturelg . . * . I 14.0 I1 1.5 5.0 - I11 1.8 4.2 1.8 - - - IV 1.125 1.5 - - 2.25 1.5 - 6.3 46.8 23-4 23.4 4.5 - - 30-65 16.2 10.3 3.0 8.025 - - to 100.0 to 50.0 - - to 70.0 - to 90.0 - to 90.0 Samples I and I1 (Vermizine elixir and coli-urinal effervescent granules) supplied by SociCtC Sample I11 (Urosolvine effervescent granules) supplied by the Nile Company for Pharma- Sample IV (Ciluryl effervescent granules) supplied by ADCO (The Arab Drug Company), Sample V (Urolithine effervescent granules) supplied by Kahira Pharmaceutical and Misr pour 1’Industrie Pharmaceutique, Cairo, Egypt.ceuticals and Chemical Industries, Cairo, Egypt. Cairo, Egypt. Chemical Industries, Cairo, Egypt. PREPARATION OF SAMPLE SOLUTIONS- For effervescent granules, transfer an amount of the sample equivalent to 100 mg of piperazine citrate (or hexahydrate, depending on the formulation) into a 100-ml calibrated flask, add about 50 ml of water, and when the effervescence has ceased, dilute to volume with water. For elixirs, dilute the appropriate volume of the sample with water to give a final concentration equivalent to 3 mg of piperazine citrate (or hexahydrate, depending on the formulation) in 100 ml of water.Dilute 3 ml of this solution to 100 ml with water. TABLE I1 COMPARISON BETWEEN THE RESULTS OF THE PROPOSED METHOD AND THE B.P. 1968 METHOD Amount of piperazine citrate (or hexahydrate) I Recovered: by proposed method & I 64.7 107.8 60-3 100.5 62.5 104.1 60.0 100.0 59.4 99.0 60.8 101.3 56.3 93.8 60.3 100.5 58-2 97.0 61.0 101.6 PFtg per cent. Recovered $ by B.P. 1968 method Standard Sample* Takenlpg addedt/pg - I 60 I1 60 I11 60 IV 60 V 60 60 - 60 - 60 - 60 - 60 - - - - - - Pg per cent. 48.0 80.0 38.4 64.0 50.1 83.5 57.6 96.0 - - - - - - - § The standard deviation is k0.97 per cent. for the proposed method. * See footnote to Table I. 7 Added as a 0.003 per cent. aqueous solution of piperazine citrate (or hexahydrate). 9 The precipitated piperazine picrate was unfilterable.Each value given is the average of three experiments.484 DESSOUKY AND ISMAIEL : COLORIMETRIC DETERMINATION OF [Analyst, VOl. 99 PROCEDURE- To 2 ml of the standard and sample solutions in separate test-tubes, add 5 ml of the acetate - citrate buffer, pH 7.5, mix well and then cool the mixtures in a water-bath at a temp- erature between 10 and 15 "C for 2 minutes. Add to each test-tube 3 ml of 1,Z-naphtho- quinone-4-sulphonate reagent, again mix well and leave them to stand for about 10 minutes. Measure the absorbance of both standard and sample solutions at a wavelength of 490 nm against a blank carried out simultaneously. A Carl Zeiss, Model M4 QII, spectrophotometer was used. Calculate the amount of piperazine citrate (or piperazine hexahydrate) as follows : x 3 x 100 = piperazine salt in sample, per cent.m/m s m2 where T and S are the absorbance values of sample and standard, respectively, m, is the amount of standard present in 2 ml, and m2 is the amount of sample present, theoretically, in 2 ml. The results are given in Table 11. a) 0.4 g 0.3 0.2 0 S B a 0- 1 0 460 500 540 580 620 660 Wave1 engt h/nm Fig. 1. Light absorbance spectrum of the colour formed RESULTS AND DISCUSSION During the study of the reaction of 1,2-naphthoquinone-4-sulphonate with some secondary amines, a red colour was observed on treating piperazine with 1,2-naphthoquinone-4-sulphon- ate at a pH between 6 and 9. 0.5 0.4 - a) f $ 0.3 2 n - a 0.2 - 5 6 7 8 9 1 0 PH Fig. 2. Optimum pH for the reaction (wavelength, 490 nm)August, 19741 PIPERAZINE IN PHARMACEUTICAL FORMULATIONS 485 A studv of the colour formed showed that the maximum absorption occurs at 490 nm (Fig.l), that ihe optimum pH for the reaction is 7.5 (Fig. 2) and that the optimum tration of the reagent is 18 mg per 10 ml (Fig. 3). concen- 0 10 18 20 30 Amount of reagentlmg 40 Fig. 3. Variation of absorbance with amount of 1,2-naphthoquinone-4-sulphonic acid, sodium salt (wavelength, 490 nm) The coloured product of the reaction started to precipitate after about 15 minutes. This time decreased with increase in the concentration of piperazine (Fig. 4). The above precipi- tation accounts for the slight differences in the calibration graphs. The addition of not only ethanol, but also methanol, propan-1-01 and propan-2-01, was tried, without success in reducing the differences.Extraction into an immiscible solvent, such as chloroform, was also tried and gave partial extraction. However, the organic layer changed to a yellowish colour. The stability of the red colour (before the formation of a red precipitate) differs according to the concentration. The most stable colour is obtained when 60 pg of piperazine salt are used (Fig. 4). 0.5 0.4 0) f 0.3 f a 2 0.2 0.1 - 1 I 1 I I I I I I I o 4 a 12 16 20 24 28 32 36 40 44 Ti me/minutes Fig. 4. Colour stability a t different concentrations of piperazine salt (wavelength, 490 nm) : A, 80 pg; B, 60 pg; and C, 100 pg Beer’s law is obeyed for amounts of piperazine citrate from 20 to 120 pg (Fig. 5), although the simultaneous use of a standard, with which to compare the sample, is found to be necessary in order to obtain accurate results, as the slope of the calibration graph differs slightly on repetition.486 DESSOUKY AND ZSMAIEL Piperazine citrate/pg Fig. 5. length, 490 nm) Calibration graph for different amounts of piperazine citrate (wave- REFERENCES 1. 2. 3. 4. 5. 6. 7. “The United States Pharmacopeia,” XVIIIth Revision, Mack Co., Easton, Pa., 1970, p. 507. “British Pharmacopoeia,” The Pharmaceutical Press, London, 1968, p. 776. Pankratz, R. E., J . Pharm. Sci., 1961, 50, 175. Perlmutter, S. H., J . Ass. Off. Agric. Chem., 1958, 41, 506. Simionovici, R., RAianu, J., and Cuculescu, V., Rev. Chim., Bucharest, 1959, 10, 105; Analyt. Maynard, W. R., jun., J , Ass. Off. Agric. Chem., 1959, 42, 610. Chemerisskaya, A. A., Zh. Analit. Khim., 1956, 11, 356; Chem. Abstr., 1956, 50, 15349’. Abstr., 1959, 6, 4154. Received April 19th, 1973 Accepted February 4 t h 1974