ELDAWY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 4,
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1 ELDAWY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 4, DRUGS, COSMETICS, FORENSIC SCIENCES Determintion of Chlorphenirmine Mlete nd Tincture Ipecc in Dosge Form by Liquid Chromtogrphy with Ultrviolet Detection MOHAMED A. ELDAWY, MOKHTAR M. MABROUK, nd FAWZY A. EL-BARBARY Tnt University, Fculty of Phrmcy, Deprtment of Phrmceuticl Chemistry, Tnt, Egypt A procedure ws developed nd vlidted for mesuring chlorphenirmine mlete nd tincture ipecc (s emetine hydrochloride) by reversed-phse liquid chromtogrphy with methnol 10mM sodium heptnesulfonte ( ) s the mobile phse; the ph ws djusted to 4 with cetic cid, nd the flow rte ws t 1.5 ml/min, with ultrviolet detection t 254 nm. Propyl prben ws used s the internl stndrd. The stndrd curves were liner (r = nd ) for both chlorphenirmine mlete nd emetine hydrochloride over the rnges of nd g/ml, respectively. The men recoveries stndrd devition were % for chlorphenirmine mlete nd % for emetine hydrochloride. The proposed method ws pplied to the determintion of chlorphenirmine mlete lone in tblet nd syrup dosge forms. The method lso ws pplied to the determintion of the emetine content of ipecc liquid extrct nd tincture ipecc; the results were compred with those of the method of the British Phrmcopoei. The proposed method ws pplied successfully to the simultneous determintion of chlorphenirmine mlete nd tincture ipecc, s emetine hydrochloride, in syrup dosge form. Both drugs nd the internl stndrd were seprted from ll interfering components in <5 min. The proposed method is simple, specific, nd economicl, when compred with other published methods tht determine ech component lone. Phrmceuticl preprtions contining both chlorphenirmine mlete nd tincture ipecc re likely to be of gret benefit in the tretment of cough nd sthmtic bronchitis; this result my be due to the ntihistminic property of chlorphenirmine, which inhibits the ction of histmine on the respirtory trct (1), in ddition to the effect of tincture ipecc s n expectornt, which lso liquefies the viscid sputum (2). Received My 16, Accepted by JM December 8, Corresponding uthor s e-mil: eldwymo@thewyout.net. Severl nlyticl methods hve been reported for the ssy of chlorphenirmine mlete. One of the colorimetric methods depends on rection with 7,7,8,8-tetrcynoquinodimethne (3). Dent nd Stewrt (4) reported fluorimetric method, using sodium coumrin-6-sulfonte s fluorimetric ion-piring regent. Midh nd Rvw (5) reported method for quntittion of subnnogrm levels of chlorphenirmine mlete in plsm by rdioimmunossy. Liquid chromtogrphy (LC) hs been widely pplied to the determintion of chlorphenirmine mlete (6 12). Determintion of emetine hydrochloride by LC requires precolumn derivtiztion with dnsyl chloride (13) or oxidtion to rubremetine to be detected by fluorescence detection (14). No nlyticl method hs been reported for the simultneous determintion of both chlorphenirmine mlete nd tincture ipecc in their dosge forms. This pper describes procedure developed nd vlidted for the determintion of chlorphenirmine mlete in its tblet nd syrup dosge forms. The method lso ws pplied to the determintion of tincture ipecc nd ipecc liquid extrct, s emetine hydrochloride, nd the results were compred with those of the method of the British Phrmcopoei (15). The method ws pplied successfully to the simultneous determintion of both chlorphenirmine mlete nd tincture ipecc in their syrup dosge form. METHOD Apprtus () Liquid chromtogrph. An isocrtic system, consisting of Wters Model 600 pump, Wters Model 486 LC spectrophotometric detector, Wters Model 499 photodiode rry detector, nd Wters Millennium dt module (Wters, Division of Millipore, Milford, MA). The LC operting conditions were s follows: flow rte, 1.5 ml/min; injection volume, 20 L; detection wvelength, 254 nm; nd temperture, C. (b) LC column. Bondpk C 18, mm, 10 m prticle size. (c) Membrne filter. For queous solutions, with pore size of 0.45 m. (d) ph meter
2 676 ELDAWY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 4, 2003 Tble 1. Quntittive prmeters for determintion of chlorphenirmine mlete nd emetine HCl by the proposed LC method Drug Linerity rnge, g/ml Intercept Slope Correltion coefficient LOD, g/ml Chlorphenirmine mlete Emetine HCl LOD = Limit of detection. Mterils () Authentic smples. Chlorphenirmine mlete, ipecc liquid extrct, nd ipecc tincture were kindly supplied by Khir Phrmceuticl nd Chemicl Industries Co. (Ciro, Egypt). (b) Emetine HCl. Sigm (St. Louis, MO). (c) Propyl prben. Sigm. (d) Phrmceuticl preprtions. Allergyl tblets, Anllerge-4 tblets, Pirfen tblets, Allergyl syrup, Pirfen syrup, Bronchistl syrup, nd Broncholse syrup were purchsed loclly. (e) Methnol. LC grde (Romil, Wterbech, UK). (f) Sodium heptnesulfonte. BDH Lbortory Supplies (Poole, UK). (g) Other regents. Anlyticl grde. Tble 2. Results of recovery experiments using the proposed method with propyl prben (10 g/ml) s the internl stndrd Added, g/ml Found, g/ml Recovery, % Chlorphenirmine mlete Men rec., % SD CV b, % 2.73 Emetine HCl Men rec., % SD 1.47 CV, % 1.48 b SD = Stndrd devition. CV = Coefficient of vrition. (h) Mobile phse. Methnol 10mM sodium heptnesulfonte ( ). Dissolve g sodium heptnesulfonte in c 100 ml distilled wter in 1000 ml volumetric flsk, nd dilute to volume with wter. Adjust ph of solution to 4 with cetic cid. Trnsfer 600 ml liquot of solution to 1000 ml volumetric flsk, nd dilute to volume with methnol. Filter solution through 0.45 m filter, nd degs for 10 min. (i) Stndrd solutions. (1) Stock solution of internl stndrd. Propyl prben in mobile phse, 100 g/ml. (2) Stock solution of chlorphenirmine mlete. 400 g/ml mobile phse. (3) Stock solution of emetine HCl. 200 g/ml mobile phse. Construction of Clibrtion Grphs Dilute liquots of chlorphenirmine mlete stock solution with mobile phse to obtin solutions hving concentrtions in the rnge of g/ml. To ech, dd pproprite liquot of internl stndrd stock solution to obtin finl propyl prben concentrtion of 10 g/ml. Dilute liquots of emetine HCl stock solution with mobile phse to obtin solutions hving concentrtions in the rnge of g/ml. To ech, dd pproprite liquot of internl stndrd stock solution to obtin finl propyl prben concentrtion of 10 g/ml. Construct clibrtion grphs by plotting pek re rtios (of drug to internl stndrd) versus drug concentrtions. Preprtion of Smple for Determintion of Emetine HCl in Ipecc Liquid Extrct nd Tincture Ipecc Trnsfer 1.0 ml ipecc liquid extrct or 10.0 ml tincture ipecc to 100 ml volumetric flsk, dilute to volume with mobile phse, nd filter. Trnsfer 1.0 ml liquot of filtrte to 10 ml volumetric flsk contining 1.0 ml internl stndrd stock solution. Dilute solution to volume with mobile phse. Determintion of Emetine HCl in Ipecc Liquid Extrct nd Tincture Ipecc Inject equl volumes (20 L) of ech stndrd nd ssy solution into liquid chromtogrph. Plot pek re rtios (of emetine to internl stndrd), nd clculte concentrtion of emetine HCl in ssy solution from clibrtion curve. The results were compred with those obtined by using the method of the British Phrmcopoei (15).
3 ELDAWY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 4, Tble 3. Recovery of chlorphenirmine mlete nd emetine HCl, from mixture with possible interfering mterils, by the proposed LC method Drug Added, g/ml Recovered, g/ml Recovery ± SD, % Chlorphenirmine mlete ± 0.38 Emetine HCl ± Ech vlue is the verge of 3 determintions; SD = stndrd devition. Preprtion of Stndrd for Determintion of Chlorphenirmine Mlete nd Tincture Ipecc, s Emetine HCl, in Synthetic Mixture Dilute liquots of stock solutions to obtin mixed stndrd solution contining chrlophenirmine mlete t 60 g/ml, emetine HCl t 25 g/ml, nd propyl prben t 10 g/ml. (Note: Some phrmceuticl preprtions contin propyl prben s preservtive; in such cses, the use of nother suitble internl stndrd is recommended.) Preprtion of Synthetic Mixture for Determintion of Chlorphenirmine Mlete nd Tincture Ipecc, s Emetine HCl Dissolve the following components in deminerlized wter, nd dilute solution to ml: chlorphenirmine mlete, 0.03 g; mmonium chloride, 1.00 g; sodium cmphosulfonte, 2.00 g; tincture ipecc, 6.80 ml; lcohol (95%), 8.00 ml; sorbitol (70%), 8.00 ml; vnillin, 0.05 g; glycerin, 7.50 g; essence tutti frutti, g; coumrin, 0.05 g; nd sugr, 60.0 g. Trnsfer 30 ml liquot of this solution to 50 ml volumetric flsk, dilute to volume with mobile phse, nd filter. Trnsfer 5.0 ml liquot of filtrte to 10 ml volumetric flsk contining 1.0 ml internl stndrd stock solution, nd dilute solution to volume with mobile phse. Determintion of Chlorphenirmine Mlete nd Tincture Ipecc, s Emetine HCl, in Synthetic Mixture Inject equl volumes (c 20 L) of ech stndrd nd ssy preprtion into liquid chromtogrph. Clculte concentrtion of ech drug in ssy preprtion with the following eqution: Cu = R / Ri Cs Rs / Ris where Cu = concentrtion of drug in ssy solution ( g/ml), R = pek re of drug in ssy solution, Ri = pek re of internl stndrd in ssy solution, Rs = pek re of drug in stndrd solution, Ris = pek re of internl stndrd in stndrd solution, nd Cs = concentrtion of drug in stndrd solution ( g/ml). Tble 4. Results of stbility study of chlorphenirmine mlete nd emetine HCl in solution over 24 h Recovered, g/ml Initil concn, 10 g/ml After 6 h After 12 h After 18 h After 24 h SD,10 g/ml RSD, % b Chlorphenirmine mlete Emetine HCl b SD = Stndrd devition. RSD = Reltive stndrd devition.
4 678 ELDAWY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 4, 2003 Tble 5. Determintion of chlorphenirmine mlete in different dosge forms by the proposed LC method Preprtion Lbel clim, g/ml Found, g/ml % of lbel clim ± SD Allergyl tblets ± Pirfen tblets ± Anllerge-4 tblets ± Allergyl syrup ± Pirfen syrup ± Ech vlue is the verge of 5 determintions; SD = stndrd devition. Determintion of Chlorphenirmine Mlete nd Tincture Ipecc in Syrup Dosge Form Trnsfer 30 ml liquot of syrup to 50 ml volumetric flsk, nd dilute to volume with mobile phse. Filter solution through 0.45 m filter. Trnsfer 5.0 ml filtrte to 10 ml volumetric flsk contining 1.0 ml internl stndrd stock solution, nd dilute solution to volume with mobile phse. Limits of Detection nd Quntittion The LOD nd LOQ were clculted ccording to the recommendtion for chromtogrphic methods, which ws published by the Interntionl Union of Pure nd Applied Chemistry (16). Accordingly, the LOD nd LOQ were 2.0 nd 5.0 g/ml, respectively, for chlorphenirmine mlete, nd 0.05 nd 0.10 g/ml, respectively, for emetine HCl. These results were verified in prcticl pplictions. Results nd Discussion Becuse there re no reported LC methods for the simultneous determintion of chlorphenirmine mlete nd tincture ipecc, we developed simple, ccurte, nd precise LC method for the simultneous determintion of both drugs without prior seprtion step. The optimum composition of the mobile phse ws found to be methnol 10mM sodium heptnesulfonte ( ). The ph ws djusted to 4 with cetic cid. In the serch for n internl stndrd, propyl prben ws found to be suitble for concomitnt determintion of chlorphenirmine mlete nd the emetine content of tincture ipecc. When the previously mentioned chromtogrphic conditions were tested, 3 well-resolved shrp peks in the chromtogrm were obtined for chlorphenirmine mlete, the internl stndrd, nd emetine with retention times of 1.33, 2.35, nd 3.16 min, respectively. Two stndrd clibrtion curves for chlorphenirmine mlete nd emetine hydrochloride were constructed by using the rtios of pek res for chlorphenirmine nd emetine t concentrtions different from tht of the internl stndrd, propyl prben, t 10 g/ml. Liner reltionships between pek re rtios nd the concentrtions of chlorphenirmine mlete nd emetine HCl were found in the rnges of nd g/ml, respectively. The good linerity of the method ws indicted by the results of the regression nlysis summrized in Tble 1. Other nlyticl prmeters for the ssessment of the vlidity of the proposed method were exmined, such s limit of detection (LOD), limit of quntittion (LOQ), ccurcy, precision, specificity, nd stbility. Figure 1. Liquid chromtogrm of chlorphenirmine mlete nd propyl prben (10 g/ml) from the ssy of Allergyl tblets.
5 ELDAWY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 4, Tble 6. Determintion of the emetine content of ipecc liquid extrct nd tincture ipecc by the proposed LC method nd the British Phrmcopoei method Emetine content ± SD, g/ml Dosge form LC method BP method Ipecc liquid extrct ± ± Tincture ipecc ± ± Ech vlue is the verge of 3 determintions (s emetine HCl); SD = stndrd devition. Accurcy The ccurcy of the proposed method ws studied by prepring synthetic mixtures contining vrious mounts of chlorphenirmine mlete nd emetine HCl nd nlyzing them by the proposed method. The men percent recoveries stndrd devition (SD) were nd for chlorphenirmine mlete nd emetine HCl, respectively. The results shown in Tble 2 indicte resonble ccurcy. Precision () Repetbility. The SD nd coefficient of vrition (CV) were clculted for 3 concentrtions from nlysis of 3 replictes t ech concentrtion. (b) Intermedite precision. Repetbility (6 determintions for one concentrtion) ws clculted on 2 different dys. In both () nd (b), the men SD nd CV were 0.45 nd 1.70%, respectively, for chlorphenirmine nd nd 0.89%, respectively, for emetine HCl. In ll cses, no CV ws >2%. Specificity The men percent recoveries of chlorphenirmine mlete nd emetine HCl were determined seprtely in mixture of them with possible interfering substnces such s cmphosulfonte nd sorbitol. The results obtined, shown in Tble 3, indicted no interference. Stbility The stbility of chlorphenirmine mlete nd emetine HCl in solution ws ssessed by determining the reltive stndrd devition (RSD) for replicte injections of the sme solution over period of time under the sme nlyticl conditions. The RSD should be less thn times the vlue for the precision obtined by nlyzing replicte injections over reltively short time period (17). The results, shown in Tble 4, indicted dequte stbility for chlorphenirmine mlete nd emetine HCl, both in solution nd during the nlysis. Sttisticl evlution of the results (nlysis of vrince), showed no significnt vritions in concentrtion with time. Figure 2. Liquid chromtogrm of emetine HCl nd propyl prben, the internl stndrd (IS; 10 g/ml) from the ssy of tincture ipecc. The proposed LC method ws pplied successfully to the determintion of chlorphenirmine mlete in some of its dosge forms. The results re shown in Tble 5. Figure 1 shows the liquid chromtogrm of chlorphenirmine mlete nd propyl prben from the ssy of Allergyl tblets. The proposed method ws pplied to the determintion of the emetine content of both ipecc liquid extrct nd tincture ipecc, nd the results were compred with those of the method of the British Phrmcopoei (15). The results re shown in Tble 6. Tble 7. Determintion of chlorphenirmine mlete nd tincture ipecc (s emetine HCl) in dosge forms by the proposed LC method % of lbel clim ± SD Product Chlorphenirmine mlete Emetine HCl Bronchistl syrup ± ± Broncholse syrup ± ± SD = Stndrd devition.
6 680 ELDAWY ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 4, 2003 Becuse this LC technique requires no pretretment, it offers cler dvntge over other LC methods. With the simple elution system, the entire nlysis tkes only 4.0 min, mking it convenient for qulity ssurnce purposes. The proposed LC method enbles simultneous determintion of both chlorphenirmine mlete nd tincture ipecc in nticough syrups, insted of seprte ssys of the individul components. Acknowledgments We thnk Khir Phrmceuticl nd Chemicl Industries Co. for its coopertion. References Figure 3. Liquid chromtogrm of chlorphenirmine mlete, propyl prben, the internl stndrd (IS; 10 g/ml), nd emetine HCl from the ssy of Bronchistl syrup. Figure 2 shows the liquid chromtogrm of emetine HCl nd propyl prben from the ssy of tincture ipecc. The proposed method ws pplied to the simultneous determintion of chlorphenirmine mlete nd tincture ipecc (for its emetine content) in syrup dosge form contining both components. The results re shown in Tble 7. Figure 3 shows the liquid chromtogrm of chlorphenirmine mlete, propyl prben, nd emetine HCl from the ssy of Bronchistl syrup; the peks re well resolved, nd the retention times re 1.28, 2.31, nd 3.16 min, respectively. Conclusions (1) Mrtindle The Extrphrmcopoei (1996) 31st Ed., The Royl Phrmceuticl Society, London, UK, p. 437 (2) Curtis, D. (1996) in Goodmn nd Gilmn, The Phrmcologicl Bsis of Therpeutics, 9th Ed., Pergmon Press, New York, NY, pp (3) AbdelKhlek, N.M., & AbdelHmid, M.E. (1985) J. Assoc. Off. Anl. Chem. 5, (4) Dent, L.L., & Stewrt, J.T. (1981) Anl. Lett. 14, (5) Midh, K.K., & Rvw, G.J. (1993) J. Phrm. Biomed. Anl. 9, (6) Chen, Y.P., Wng, P., & Chng, B.L. (1999) Yowu Shipin Fenxi 7, (7) Zhng, S.L. (1999) Yowu Shipin Fenxi 19, (8) Hgink, J., & Mtsung, H. (1999) J. Chromtogr. A 830, (9) Erk, N., & Krtl, M. (1998) Frmco 53, (10) Hgink, J., & Mtsung, H. (1999) Chromtogrphy 19, (11) Stephni, R., & Csre, V. (1998) J. Chromtogr. A 813, (12) Aldeeb, O.A., & Fod, N.H. (1997) J. Liq. Chromtogr. Relt. Technol. 20, (13) Frei, R.W., Snti, W., & Thoms, M. (1976) J. Chromtogr. 116, (14) Bnnister, S.J., & Stevens, J. (1979) J. Chromtogr. 176, (15) British Phrmcopoei (1998) Her Mjesty s Sttionery Office, London, UK, p. 176 (16) Crr, G., & Whlich, J. (1994) in Interntionl Phrmceuticl Product Registrtion, C. Anthony & R. Brin (Eds), p. 247 (17) Szepesi, G., Gzdg, M., & Mihlyfi, K. (1989) J. Chromtogr. 464,
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