Acta Chim. Slov. 2004, 51, 283-291. 283 Scientific Paper FLUORIMETRIC DETERMINATION OF CARBOCISTEINE AND ETHIONAMIDE IN DRUG FORMULATION Mohamed L Walash,* Amina M. El-Brashy, Mohamed E.-S. Metwally, and Amina A. Abdelal Analytical Chemistry Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt Received 05-08-2003 Abstract A highly sensitive and specific fluorimetric method was developed for the determination of carbocisteine and ethionamide in their dosage forms. The proposed method based on the reaction of carbocisteine and ethionamide with roth's reagent (o-phthaldehyde) to get a highly fluorescent isoindole product emits strong fluorescence at 431 nm and 424 nm after excitation at 329 nm and 339 nm for carbocisteine and ethionamide, respectively. The thiol group present in these compounds is responsible for the formation of highly fluorescent complexes of improved stability and enhanced fluorescence. The different experimental parameters affecting the intensity of the fluorescence were carefully studied and incorporated into the procedure. Under the described conditions, the method was applicable over the concentration range of 0.05-0.9 µg/mL and 0.25-2.5 µg/mL with detection limits of 5 ng/mL and 26 ng/mL for carbocisteine and ethionamide, respectively. The proposed method was successfully applied for determination of carbocisteine and ethionamide in their dosage forms. Key words: flourimetry, o-phthaldehyde, carbocisteine, ethionamide, dosage forms Introduction The importance of carbocisteine [1] and ethionamide [II] is due to their widespread use and different pharmacological effects. Carbocisteine (S-carboxymethyl-L-cysteine) is a mucolytic drug used for treatment of disorders of the respiratory tract associated with excessive mucus. Ethionamide (2-ethyl-thioisonicotinamide) is a tuberculostatic agent used in treatment of isoniazid-resistant tuberculosis.1_2 ^-Nk ^Et H JSH2 f HOOC. .S. X ^v ^^ ^-^ COOH CSNH2 [1] [H] The published methods for the determination of these drugs include titrimetry,3"4 spectrophotometry,5-9 electro-analysis,10 and chromatography.11-16 The above mentioned M. I. Walash, A. M. El-Brashy, M. E.-S. Metwally, A. A. Abdelal: Fluorimetric Determination... 284 Acta Chim. Slov. 2004, 51, 283-291. methods were either not sufficiently sensitive or were tedious and require highly sophisticated instrumentation. Therefore, there was stili a need for a much more sensitive and simple method for the determination of carbocisteine and ethionamide. Reviewing the literature revealed that no fluorimetric methods were reported for these drugs. As for o-phthaldehyde, it has been recently used for determination of different thiols,17 prazosin hydrochloride,18 acidic polysaccharides,19 and amino acids.20"21 The aim of the present work is to study the reaction of o-phthaldehyde and the primary amino group in presence of thiol group in these thiocompounds in an attempt to develop a highly sensitive fluorimetric method to be used for their determination in their dosage forms. Experimental Equipment ARF-1501 Shimadzu spectrofluorophotometer with xenon lamp. A 1 cm quartz celi was used for ali measurements. Reagents and Materials Ali reagents used were of analytical reagent grade and the water was always double distilled water. Carbocisteine was offered by Amyria Pharmaceutical Industries, Egypt. Ethionamide was offered by Alexandria Theraplix Company, Egypt. The purities of these drugs were determined by applying the official methods.3 Stock solutions of the studied drugs were prepared by dissolving 5 mg of carbocisteine and ethionamide in 3 mL of 0.2 M NaOH and 3 mL of 5 N HC1 respectively then completed to 100 mL with distilled water. Other concentrations were prepared by dilution with distilled water. 0.05% o-phthaldehyde (Sigma, UK) was freshly daily prepared in methanol. Aqueous solutions of 0.2 M NaOH (BDH, UK) and 5N HC1 (Prolabo) were prepared. Procedures Construction of calibration graphs Transfer aliquots of carbocisteine or ethionamide equivalent to 0.05-0.9 µg/mL or 0.25-2.5 µg/mL respectively into a series of 10 mL volumetric flasks. 0.8 mL or 1.5 mL M. I. Walash, A. M. El-Brashy, M. E.-S. Metwally, A. A. Abdelal: Fluorimetric Determination... Acta Chim. Slov. 2004, 51, 283-291. 285 of 0.05% o-phthaldehyde was added to the 2 drugs respectively followed by 1 mL of 0.2M NaOH. The volume was adjusted to the mark with distilled water. The fluorescence was measured at X Em. 431 or 424 nm after excitation at 329 nm or 339 nm for carbocisteine or ethionamide after 45 min, at ambient temperature, respectively. A blank reagent was prepared simultaneously. Procedure for the dosage forms An accurately weighed quantity of the mixed contents of 10 pulverized tablets or measured volume of the syrup equivalent to 5 mg of the drug was transferred into a 100 mL volumetric flask and made up to the mark with distilled water. 3 mL of 0.2 M NaOH or 3 mL of 5N HC1 were firstly added in čase of Rhinathiol® syrup or Trecator® tablets, respectively. The contents of the flask were sonicated for 5 min, filtered, and the above procedure was completed as before. The amount of carbocisteine or ethionamide was calculated either from a previously prepared calibration graph or using the regression equation. Results and Discussion Carbocisteine and ethionamide are sulfur compounds containing both sulfur and primary amino groups that react with o-phthaldehyde giving a highly fluorescent isoindole products22 at 431/329 nm and 424/339 nm (kemJKx.) for these two drugs (figures 1 and 2). i -i i i i------------------------------------------- 250 600 Wavelength (nm) Figure 1. Fluorescence spectra of Carbocisteine (0.9 ug/mL) after reaction with o-phfhaldehyde o-phthaldehyde at Xex. = 329 nm (A), ?iem = 431 nm (B). M. I. Walash, A. M. El-Brashy, M. E.-S. Merwally, A. A. Abdelal: Fluorimetric Determination... 286 Acta Chim. Slov. 2004, 51, 283-291. 1000 250 600 Wavelength (nm) Figure 2. Fluorescence spectra of Ethionamide (2.5 µg/mL) after reaction with o-phthaldehyde o-phthaldehyde at ?ex = 339 nm (A), ?em = 424 nm (B). The sulfur group present in these compounds is responsible for formation of highly fluorescent complexes of improved stability and enhanced fluorescence. The effect of temperature on the fluorescence intensity was studied in the range 30-80 °C. It was observed that the reaction not quantitized at higher temperature. Optimization of the reaction conditions Effect of o-pthaldehyde From Figure 3, it was found that increasing the volume of 0.05% o-phthaldehyde up to 0.8 mL and 1.5 mL for carbocisteine and ethionamide respectively, would increase the fluorescence intensity of the reaction and after that the fluorescence intensity decreases. 240- 200- 160- 120- 80- 40- 0- 0.0 Ethionamide (2 µg/mL) Carbocisteine (0.5 µg/mL) 2.4 0.4 0.8 1.2 1.6 2.0 0.05% o-phthaldetyde (mL) Figure 3. Effect of o-pthaldehyde on the Flourescence intensity. M. I. Walash, A. M. El-Brashy, M. E.-S. Metwally, A. A. Abdelal: Fluorimetric Determination... Acta Chim. Slov. 2004, 51, 283-291. 287 Effect of NaOH From Figure 4, increasing the volume of 0.2M NaOH up to 1 mL for both compounds would increase the fluorescence intensity of the product and after that the fluorescence intensity decreases. Relative Flourescence 400 300 - 200 - 100 - 0 o Carbocisteine (0.5 µ g/mL) Ethionamide ( 2 µg/mL) 4 5 123 0.2 M NaOH Volume (mL) Figure 4. Efect of NaOH volume on the fluorescence intensity. Effect of time The fluorescence intensity of the products of the two studied drugs increases with time up to 45 min (at room temperature), after that it remains stable for more than one hour Figure 5. 400- 300- 200- 100- 0-f 1 10 I 20 Carbocisteine (0.5 µg/mL) Ethionamide (2 µg/mL) 1 30 1 40 i— 50 1 60 1 70 1 80 Time (min) Figure 5. Effect of reaction tirne on the reaction between the studied compounds and o-phthaldehyde. 0 M. I. Walash, A. M. El-Brashy, M. E.-S. Metwally, A. A. Abdelal: Fluorimetric Determination... 288 Acta Chim. Slov. 2004, 51, 283-291. Calibration graphs After optimizing the conditions, it was found that the relation between relative intensity (RI) and final concentration of carbocisteine and ethionamide was rectilinear over the range 0.05-0.9 µglmL and 0.25-2.5 µglmL with detection limit of 5 ng/mL and 26 ng/mL for the two drugs respectively Figure 6. 500 400 300 200 100 -•- Carbocisteine -o- Ethionamide 01 23 Concentration ( µg/mL) Figure 6. Calibration Curve of Carbocisteine and Ethionamide after reaction with 0.05% o-phthaldehyde. The linear regression analysis of the results are: RI = -0.983+247.877 C (r = 0.9999) for carbocisteine and RI = 1.518+168.134 C (r = 0.9999) for ethionamide. Where: C=fmal concentration in µg/mL, RI = relative fluorescence intensity (fluorescence intensity of the product - fluorescence intensity of the o-phthaldehyde) and r = correlation coefficient. The % recoveries of these studied drugs compared with that obtained by the official methods3 were given in table 1. These official methods recommends non-aqueous titration for the two drugs. Statistical analysis of the results obtained by the proposed and official methods3 revealed that no significant difference between the two methods regarding the accuracy and precision as indicated by the F-test and študent’s t-test.23 The proposed method was successfully applied for the determination of the studied sulfur drugs in their dosage forms, as shown in Table 2. 0 M. I. Walash, A. M. El-Brashy, M. E.-S. Metwally, A. A. Abdelal: Fluorimetric Determination... Acta Chim. Slov. 2004, 51, 283-291. 289 Table 1. Fluorimetric determination of carbocisteine and ethionamide by o-phthaldehyde in pure form. Compound Proposed method Official method3 Taken Found Rec/ Taken Rec.* (µg/mL) (µg/mL) % (mg) % Carbocisteine 0.05 0.049 98.00 0.1 0.100 100.00 100 100.36 0.2 0.198 99.00 150 99.16 0.4 0.399 99.75 0.5 0.496 99.20 200 99.46 0.6 0.601 100.16 0.8 0.802 100.25 0.9 0.899 99.89 Mean ± S. D. 99.53+0.76 99.66 + 0.624 t-test 0.635 2.262" F-test 1.483 19.36 Ethionamide 0.25 0.247 98.80 0.5 0.502 100.40 100 99.67 1 1.002 100.2 1.5 1.501 100.06 150 98.56 Mean ± S. D. t-test F-test 2.5 1.995 2.501 99.75 100.04 99.88+ 0.568 0.813 3.17 200 99.60+ 1.012 2.365** 5.79 100.58 * Each result is the average of three separate experiments. " The values are the tabulated študent’s t-test and Variance ration test (at P = 0.05).23 The results obtained by the proposed method are compared with that of the reference methods.5'6 The reference methods recommends spectrophotometric methods for the two drugs, where the absorbance methanolic solution of ethionamide was measured at 290 nm5 and complexation of carbocisteine with nickel and measuring the absorbance of the formed complex at 259 nm.6 Conclusions The proposed method was simple, accurate, precise, reproducible, highly sensitive, and relatively selective compared to the official method. 2 M. I. Walash, A. M. El-Brashy, M. E.-S. Metwally, A. A. Abdelal: Fluorimetric Determination... 290 Acta Chim. Slov. 2004, 51, 283-291. Table 2. Fluorimetric determination of carbocisteine and ethionamide by o-phthaldehyde in their dosage forms. Proposed method Reference method Compound Taken Found Rec/ Taken Rec.* (µg/mL) (µg/mL) % % 1) Carbocisteine®syrup 6 (Carbocisteine, 2g/100mL) 0.4 0.406 101.63 10 µg/mL 101.20 0.6 0.605 100.95 20 µg/mL 100.36 0.8 0.808 101.12 30 µg/mL 40 µg/mL 50 µg/mL 98.57 98.93 101.02 Mean ± S. D. 101.23 + 0.354 99.95+ 1.189 2) Trecator®tablets 5 (Ethionamide, 250 50 mg 98.92 mg/tablet) 0.5 0.494 98.84 1 1,01 100.59 100 mg 99.81 2.5 2.488 99.53 150 mg 98.14 Mean ± S. D. 99.65 ± 0.881 98.96 + 0.836 The results are the average of 6 separate determinations. 1) Amyria Pharmaceutical Company, Egypt. 2) Alexandria Theraplix Company, Egypt. Furthermore, the proposed method does not require elaboration of procedures which are usually associated with chromatographic methods. The proposed method could be applied successfully for determination of carbocisteine and ethionamide in pure form as well as in different dosage forms. Our method allows the determination of lower amounts of the studied compounds than those detectable by the official methods that require non-aqueous titration for carbocisteine and ethionamide.3 The proposed method was found to be with detection limits of 5 ng/mL and 26 ng/mL and quantification limits of 0.05 ug/mL and 0.25 ug/mL for carbocisteine and ethionamide, respectively. References 1. Martindale (The Extra Pharmacopoeia)-32 Ed., The Pharmaceutical Press; London, 1999, pp 1056-1057, p 208. 2. Merck Index, 12 Ed., by Merck & Co. Inc.; White House Station, N.J., 1995, p 294, p 638. 3. British Pharmacopoeia, London, 1998, pp 249-250, pp 553-554. 4. H. Sikorska-Tomicka, H. Sobolewska, Chem. Anal. (Warsaw) 1986, 31, 453-457. 5. The United States Pharmacopoeia XXII. National Formulary XVII; Rockville, MD, USP convention, Inc., 2000. 6. 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Spectrom. 1997, 32, 388–394. 17. F. Dai, V. P. Burkert, H. N. Singh, W. L. Hinze, Micochem. J. 1997, 57, 166–198. 18. P. Parimoo, P. V. Ballimane, Indian Drugs 1997, 34, 21–23. 19. M. Kobayashi, Y. Chiba, Anal. Biochem. 1994, 219, 189–194. 20. T. Miura, Yakugaku-Kenkya-Noshinpo 1991, 7, 87–97. 21. K. Yokotsuka, I. Kato, J. C. Ogbonna, Y. Amano, J. Ferment. Bioeng. 1989, 68, 222–224. 22. S. S. Simons, D. F. Johnson, J. Am. Chem. Soc. 1976, 98, 7098. 23. R. Caulcut, R. Boddy, “Statistics for Analytical Chemists”; Chapman and Hall, 1983. Povzetek Razvili smo občutljiv in specifičen fluorimetrični postopek za določanje karbocisteina in etionamida v farmacevtskih pripravkih. Predlagani postopek temelji na reakcijah karbocisteina in etionamida z o-ftalaldehidom, pri čemer nastane fluorescirajoči produkt izoindol, ki pri vzbujevalni valovni dolžini 329 nm fluorescira pri 421 nm za karbocistein oziroma pri 424 nm za etionamid. V le-teh je za tvorbo sorazmerno stabilnih fluorescirajočih spojin pomembna tiolna skupina. Preučili smo različne eksperimentalne parametre, ki lahko vplivajo na intenzivnost fluorescence produkta. Z optimizacijo smo razvili metodo, ki daje uporabne rezultate v koncentracijskem območju 0,05-0,9 µg/mL za karbocistein (meja zaznavnosti 5 ng/mL) oziroma 0,25-2,5 µg/mL za etionamid (meja zaznavnosti 26 ng/mL). Predlagano metodo smo uporabili za določanje karbocisteina in etionamida v farmacevtskih pripravkih. M. I. Walash, A. M. El-Brashy, M. E.-S. Metwally, A. A. Abdelal: Fluorimetric Determination...