untitled ISSN 215 Spectro pyridox Neven Ma Maha Moh Pharmaceutical * Corresponding Tel.: +2.0122.50 ARTICLE IN DOI: 10.5155/e Received: 24 Oc Received in rev Accepted: 22 No Published onlin Printed: 31 Mar KEYWORDS Cyclizine HCl Dosage forms Meclizine HCl Pyridoxine HCl Spectrophotom Determination 1. Introduct Pyridoxi dine dimeth soluble vita carbohydrat used in the t and symptom [2]. Cyclizin diphenyl‐4‐m derivative an activity, it i nausea and operative na chemically k methylpheny zine derivat properties. I nausea and including mo Reviewin been publish binary mixtu 53‐2249 (Print) photome xine hydro agdy Habib * hamed Abde l Analytical Chemis g author at: Pharm 078693. Fax: +2.82.2 FORMATION eurjchem.7.1.30‐36 ctober 2015 vised form: 21 Nov ovember 2015 ne: 31 March 2016 rch 2016 S metric methods of pyridoxine hydr tion ne hydrochlor anol hydrochlo amin which is e and fat meta treatment of m ms associated e hydrochlorid methylpiperazin nd sedating an s used as an d vomiting in ausea and vomi known as 1‐[(4‐ yl)methyl]piper tive with anti‐m t is also used i vomiting assoc otion sickness [ ng the literatur hed for determ ures such as s / ISSN 2153‐225 ht Europ tric meth ochloride *, Nada Saye elrahman an stry Department, Fa aceutical Analytica 2317950. E‐mail ad 6.1350 ember 2015 rochloride ride, 5‐hydroxy oride (Figure 1 involved mai abolism. Pyrido any disorders i with the prem de is chemical ne (Figure 1) [ ntihistamine w antiemetic in ncluding moti iting [2]. Mecliz ‐chlorophenyl)p razine (Figure muscarinic and in the preventi ciated with a v 2]. re in hand, diff mination of PY second derivat European Journ Europ 57 (Online)  20 ttp://dx.doi.org pean Jo Journal web hods for an ed Abdelwha nd Nourudin aculty of Pharmacy al Chemistry Depar ddress: neeeven_ma ABSTRACT Two accurate developed an hydrochloride synthetic tern been determi dividing the interference f (AUC) has be measuring the division spect centering of ratio spectra mean centere and MEH, res the cited drug with the rep difference bet Cite this: Eur. y‐6‐methyl‐3,4 1) [1]. It is a w inly in amino oxine has also including depre menstrual synd lly designated [1]. It is a piper with anti‐ musc the manageme on sickness, zine hydrochlor phenyl methyl] 1) [1]. It is a p d moderate sed ion and treatm variety of cond ferent methods YH and CYH in tive of ratio sp al of Chemistry 7 pean Journal of C 016 Atlanta Pub /10.5155/eurjch ournal bpage: www. nalysis of ab, n Wageih Ali y, Beni‐Suef Univers rtment, Faculty of P agdy@yahoo.com ( e, precise, sens nd validated for e (CYH) and mec nary mixtures. I ined by measur ternary mixtur from CYH and en used for calc e area under the trum and after s ratio spectra sp in two success ed second ratio pectively. The p gs in their phar ported methods tween them rega . J. Chem. 2016, pyri‐ water‐ acid, been ession drome as 1‐ razine carinic ent of post‐ ride is ‐4[(3‐ ipera‐ dative ent of ditions s have their pectra (2D 1) s Wh by a Figu hydr 7 (1) (2016) 30‐ Chemistry lishing House LL hem.7.1.30‐36.1 of Che .eurjchem.co f different i sity, Beni‐Suef, 625 Pharmacy, Beni‐Sue (N.M. Habib). sitive and sele r determination clizine hydrochl In modified area ring amplitude v re by standard MEH) while are culating CYH and e curve in the ra subtraction of th pectrophotometr ive steps have spectra amplitu roposed method rmaceutical form s using student arding both accu 7(1), 30‐36 D) spectrophot spectrophotom hile the binary a RP‐HPLC met ure 1. Chemical s rochloride (b) and ‐36 LC ‐ All rights re 1350 emistry m t dosage f 14, Egypt ef University, Beni‐S ective spectroph n of pyridoxine oride (MEH) eit a under the cur value of the pla spectrum of 2 ea under the cu d MEH concentr ange of 215‐228 he constant valu ric method (MC been used for m udes at 228.8, 26 ds were success mulations. Also t's‐t and F‐tests uracy and precis tometric metho metric method a mixture a long hod [4]. HO HO O N N N N structure of pyrid meclizine hydroch served ‐ Printed y forms con Suef, 62514, Egypt. hotometric met e hydrochloride ther in their bina rve method (Me ateau at 283 nm 20 µg/mL of P urve spectropho rations in the te and 230‐243 nm ue at 283 nm. M CR) at which th measuring the a 62.0 and 270.8 fully applied for they were statis s and there w ion. od, partial least and a RP‐HPLC g with caffeine N H HCl . HCl N Cl . doxine hydrochlor hloride (c). d in the USA ntaining . thods have bee (PYH), cyclizin ary mixtures or ethod I), PYH ha m (obtained aft PYH at which n otometric metho ernary mixture b m in the obtaine Method II is mea he mean centere amplitudes of th nm for PYH, CY r determination stically compare as no significa t squares (PLS‐ C methods [3]. were analyzed (a) (b) . 2HCl (c) ride (a), cyclizine en ne in as er no od by ed an ed he YH of ed nt ‐ . d e Habib et al. / European Journal of Chemistry 7 (1) (2016) 30‐36 31 On the other hand PYH and MEH binary mixture was resolved by different spectrophotometric methods such as simultaneous equation method (using the absorbance at 231 and 220 nm) and formation of ion pair complex with FeCl3 spectrophotometric method [5]. Also different RP‐HPLC methods were reported for determination of PYH and MEH mixture [6‐8], this mixture was also analyzed using a TLC‐ densitometric method [8]. The ternary mixture containing PYH, MEH and caffeine was analyzed by the extension of Vierordt's spectrophotometric method [9]. Moreover, the ternary mixture of PYH, MEH and buclizine was determined either in pharmaceutical formulation or in human serum by a HPLC method [10]. From the previous literature review, no reported method has been developed for determination of the three components. So, the goal of the present work is to develop and validate two spectrophotometric methods for determination of ternary mixture containing PYH, CYH and MEH which will be time and money saving that is important factors in quality control drug analysis. The developed methods are selective, accurate, precise, and could be successfully applied to their pharmaceutical formulations. 2. Experimental 2.1. Instruments A double beam UV‐Visible spectrophotometer (SHIMADZU, Japan), model UV‐1601 PC with 1 cm path length, quartz cell is used and connected to IBM compatible computer. The spectrophotometer is operated by using UVPC personal spectroscopy software version 3.7. Matlab® version 2007b used for the proposed MCR method. 2.2. Samples 2.2.1. Pure samples Pyridoxine HCl and cyclizine HCl were kindly supplied by AMOUN Pharmaceutical Company (El Obour city, Cairo, Egypt). Their purity was found to be 99.75 and 99.40%, respectively, according to manufacturer certificates. While meclizine HCl was supplied by SIGMA Pharmaceutical Company (Zone 1, Moubarak Industrial City, Quesna, Menoufia, Egypt). Its purity was 99.65% according to manufacturer certificate. 2.2.2. Marketed samples Emetrex® tablets (Batch No. 20766) manufactured by AMOUN Pharmaceutical Company labeled to contain 30 mg of PYH and 50 mg of CYH per tablet. Dizerest B6® tablets (Batch No. 30248) manufactured by SIGMA Pharmaceutical Company labeled to contain 50 mg PYH and 25 mg of MEH. 2.3. Chemicals and solvents All chemicals and solvents used throughout this work were of analytical grade, and the solvents were of analytical grade and were used without further purification, methanol (Sigma‐Aldrish, Chemie GmbH, Germany) and hydrochloric acid (El‐Nasr pharmaceutical Chemicals Co., Abu‐Zabaal, Cairo, Egypt). 2.4. Solutions Stock standard solutions of PYH, CYH and MEH were prepared in methanol in the concentration of 1 mg/mL. Working standard solutions of PYH, CYH and MEH (0.1 mg/mL) were prepared by suitable dilutions of their respective stock solutions using 0.05 N HCl solution. 2.5. Laboratory prepared mixtures Different mixtures containing different ratios of PYH, CYH and MEH were prepared using their respective working solutions (0.1 mg/mL), including the ratio of their marketed formulations and using 0.05 N HCl as a solvent. 2.6. Procedure 2.6.1. Spectral characteristics of PYH, CYH and MEH Zero order absorption spectra of 5 μg/mL each of PYH, CYH and MEH were recorded from 200 to 400 nm using 0.05N HCl as a solvent. 2.6.2. Construction of calibration curves 2.6.2.1. Modified area under curve method (Method I) Accurate aliquots equivalent to 50‐400, 40‐220 and 30‐ 200 µg of PYH, CYH and MEH, respectively, were transferred separately from their respective working standard solutions (100 µg/mL) into three separate series of 10 mL volumetric flasks. The volume was completed to the mark with 0.05 N HCl solution to obtain the final concentration ranges of each one. The absorption spectra of the prepared solutions were measured in the range of 200‐400 nm. For determination of PYH, the obtained spectra were divided by standard spectrum of 20 µg/mL of PYH and then the amplitude value of the plateau at 283 nm were recorded where no interference from CYH and MEH was found. Then the calibration graph was constructed relating the amplitudes values to the corresponding concentrations of PYH. For CYH and MEH the area under curve method (AUC) was applied. Where the area under the curve in the range of 215‐228 (λ1‐λ2) and 230‐243 nm (λ3‐λ4) were recorded for the prepared solutions of pure CYH and MEH. The absorptivity (Y) value was then calculated for each component where Y = the recorded area under the curve of the component from (215‐228 nm or 230 243 nm)/concentration of the component in µg/mL. The concentrations of the components in the prepared solutions were determined by using Cramer's rule and matrices according to the following equations: A1 = Yx1Cx + Yz1Cz (λ1‐λ2) (1) A2 = Yx2Cx + Yz2Cz (λ3‐λ4) (2) A1, A2 are the area under the curve in the range of 215‐228 and 230‐243 nm, respectively. Yx1, Yx2 are the absorptivity values of CYH at (λ1‐λ2) and (λ3‐λ4), respectively. Yz1, Yz2 are the absorptivity values of MEH at (λ1‐λ2) and (λ3‐λ4), respectively. Cx and Cz are the concentration of CYH and MEH, respectively. 2.6.2.2. Mean centering of ratio spectra spectrophotometric method (MCR) (Method II) Accurately measured aliquots equivalent to 50‐400, 10‐ 220 and 20‐200 µg of PYH, CYH and MEH, respectively, were separately transferred from their working standard solution (100 µg/mL) into three separate sets of 10 mL volumetric flasks. The volume was then completed with 0.05 N HCl. The absorption spectra of the prepared solutions were measured in the range of 200‐400 nm. In order to determine PYH, 20 µg/mL of CYH was used as a divisor where the stored spectra of PYH were divided by it to obtain the first ratio spectra then these spectra were mean centered. These vectors were then divided by the mean centered ratio of αMEH /αCYH and the mean centering of the second ratio spectra were then obtained [11]. Also the recorded spectra of CYH were divided by the spectrum of PYH 32 (20 µg/mL centered, th divided by t obtain the centered. By divided by th obtained rat were divided and the seco The mea 228.8, 26.0 respectively, correspondin calibration c were compu 2.6.3. Analys The inst curves for ea spectra of d previously c calculating P 2.6.4. Applic and Dizeres Ten tabl separately g amount of E (containing 3 50 mg of P Dizerest B6® The accu to two 50 m and the prep cooled well (containing solutions (0. the previous HCl solution. Different mentioned p Emetrex® an of PYH, CY previously co recoveries w 2.6.5. Applic When p different kn and MEH (8 pharmaceuti previously m 3. Result an The com for safely t vomiting dur method for ternary mixt highly select determinatio combined do The abs overlap whi mixture mo ratio spectr severe overla then the obt ese vectors (m the mean cent second ratio y the same way, he standard spe tio spectra we d by the mean c nd ratio spectra an centered val 2 and 270.8 , were record ng concentrat curves were con ted. sis of laborator tructions given ach method we different labor computed regr PYH, CYH and M cation to pharm t B6® tablets) lets each of E grinded, mixe Emetrex® pow 30 mg PYH) an PYH (containin ® powder. urately weighed mL volumetric f pared solution and filtered either 1 µg/mL .1 mg/mL) wer sly prepared sa . t dilutions were procedure for nd Dizerest B6® YH and MEH omputed regre were then calcul cation of stand performing th own concentra 0‐120%) were ical formulation mentioned meth d discussion mbination of PY treatment and ring pregnancy determination ture. So this w tive and precis on of the thr osage forms. orption spectr ch makes dete re difficult, Fi rophotometric ap of the spectr H tained ratio s mean centered tered (MC) rat spectra which , the recorded s ectrum of 20 µg ere mean cent centered ratio a were then me lues of the sec nm for PYH ed and then tion of each nstructed and r ry prepared m under constru ere followed bu atory prepared ression equati MEH concentrati maceutical form Emetrex® and ed and accura wder equivalent d weighed an a ng 25 mg MEH d powders wer flasks. 50 mL m ns were sonicat to be used L PYH or CYH) re prepared by ample stock so e prepared and each method ® prepared sam were then c ssion equations lated. dard addition te he standard a ations of pure e accurately ad n samples befo hods. H with CYH or d /or prevent y [12]. Till now n of PYH, CYH work seeks to d e methods of a ree drugs tog a of PYH, CYH ermination of e gure 2. Deriva methods were ra of the three d Habib et al. / Eu spectra were ratio spectra) tio of (αMEH/αP h were then spectra of MEH g/mL of PYH an ered. These ve (MCR) of (αCYH/ ean centered. cond ratio spec H, CYH and plotted agains component. regression equa mixtures uction of calibr ut using the sca d mixtures an ons were use ions. mulation (Eme Dizerest B6® ately weighted t to 50 mg of amount equival H) was taken re separately m methanol was a ted for 15 min as stock solu ). Working sta suitable dilutio lutions using 0 d then the prev was carried o mples. Concentra calculated from s and the perce echnique addition techn standard PYH ded to the pre re proceeding MEH has been ion of nausea there is no rep H and MYH in develop and va analysis for acc ether and in H and MEH sh each of them i ative and deriv e tried but d drugs, these me ropean Journal o mean were PYH) to mean H were nd the ectors /αPYH) ctra at MEH, st the Their ations ration anned nd the ed for etrex® were d. An f CYH lent to from moved added n then utions ndard ons of 0.05 N iously out on ations m the entage nique, H, CYH epared in the n used a and ported their alidate curate their howed in the vative ue to ethods faile mod spe and inte Figu hydr (…..) solv 3.1. spe spe sele resu con ana and con and con sele was und is a of t exa and sele etha rega HCl met 3.2. 280 trie resu met pea divi inte pea con cali was Y = whe con of Chemistry 7 (1 ed to resolve dified area und ectra method to d MEH concent erference. ure 2. Zero order rochloride (___), cy ) and mixture con vent. . Method devel As it is importa ectrophotometr ectra of the st ectivity had to ults [13]. These Effect of div ncentration has alytical parame d intercepts o ncentrations of d 20 µg/mL for ncentration of th ectivity therefo s used as diviso der the curve m a critical step in the method [1 amined and it w d 230‐243 nm ectivity of CYH a Effect of solven anol, acetonitr arding sensitiv l was the best s thods. . Modified area As shown in F 0 nm at which n ed to determine ults were obta thod and for de ak amplitude w ision by stand erference from ak amplitude ncentration of ibration curve s found to be: 0.0493x + 0.02 ere Y is the ncentration in µ ) (2016) 30‐36 their spectra der curve metho o the spectral d trations could r absorption spect yclizine hydrochlo ntaining 5 µg/mL o lopment and op ant to develop ic methods t tudied drugs, be studied and e factors include visor and its a great effect o ters such as co of the calibrat PYH, CYH and r each). It was he divisors had re 20 µg/mL o or for all the de method (AUC), s n method optim 14]. So differe was found that w were the mo and MEH, respe nt: Different so rile, water, 0.05 vity and selectiv olvent for the s a under curve m igure 2, PYH ha no calibration fr e it in zero ord ined. In order etermination o was measured ard spectrum CYH or MEH was plotted PYH in the ra was construct 237 r = 0.9998 peak amplitu µg/mL and r is t al overlap. By od and mean ce data of the mixt be determine tra of 5 µg/mL e oride (‐‐‐‐), mecliz of each (_._._) usin ptimization and validate a h to resolve th so factors aff d optimized to o e: concentration: on the method orrelation coef tion equations d MEH were tes s observed tha d a great effect of each of PYH, eveloped metho election of wav mization as it aff ent wavelength wavelength ran st suitable ran ectively. olvents were tr 5 N HCl and 0 vity, it was fou suggested spect method (metho as an extended rom CYH and M er spectra at 2 to enhance se f PYH in the te d at 283 nm of 20 µg/mL P was observed, versus the ange of 5‐40 µ ted. The regres ude at 283 n he correlation c y applying the ntering of ratio ture, PYH, CYH d without any ach of pyridoxine zine hydrochloride ng 0.05 N HCl as a highly selective e overlapping fecting method obtain the best : The divisor selectivity and fficients, slopes s. So different sted (5, 10, 15 t changing the on the method CYH and MEH ods. In the area velength ranges fects selectivity h ranges were nges of 215‐228 nges regarding ried (methanol, 0.05 N NaOH), und that 0.05 N trophotometric od I) spectrum over MEH. Hence, we 85 nm but bad lectivity of the ernary mixture, obtained after PYH where no , Figure 3. The corresponding µg/mL and the ssion equation (3) nm, C is the coefficient. e o H y e e a e g d t r d s t 5 e d H a s y e 8 g , , N c r e d e , r o e g e n e Table 1. Assay ClMixture no PY 101 9.2 303 204 355 126 Mean±SD a Average of thr In order of the plate spectrum ob standard spe area under th Figure 3. Divis (___) and 20 hydrochloride hydrochloride hydrochloride a Figure 4. The hydrochloride absorption spec The area wavelength λ4) of CYH calculated. F spectra in th 228 nm (λ3‐λ also calculat were calcula concentratio Cramer’s ru Concentratio sample solu equations: A1=1.2308 C A2=0.6094 C where CCYH a µg/mL, respe results for the det laimed taken (µg/ CYH YH 6.00 0.00 15.00 00 18.00 0.00 10.00 0.00 15.00 5.00 18.00 2.00 ree determinations to determine C eau at 283 nm btained after d ectrum of 20 µg he curve metho sion spectra of 10 µg/mL of cycliz (…..) and a mixt (_._._.) using stan as a divisor. e selected area un (‐‐‐‐) and mecli ctra and in a mixtu a under curve ranges 215‐22 in the concent For MEH, area he wavelength r λ4) in the conce ted. The absorp ated at each w ons of CYH and ule and matr on of the two tion were calc CYH + 1.0802 CM CYH + 1.6324 CM and CMEH are the ectively. 1.2308 Habib et a termination of PYH Rec/mL) Me PYMEH 99.5.00 10010.00 99.12.00 10010.00 99.15.00 99.6.00 99. s. CYH and MEH, m is subtracted division of the g/mL of PYH Fi od was applied. 0 µg/mL each of p zine hydrochlorid ture containing 1 ndard spectrum o nder curve for de izine hydrochlori ure (‐.‐.‐) in the sub of the absorp 8 nm (λ1‐λ2) a tration range a under curve ranges 215‐228 entration range ptivity ‘Y’ valu wavelength ran MEH can be o rices in Equa drugs in mixe culated accordi MEH at 215‐228 n MEH at 230‐243 n e concentration 8 and 0.6094 ar al. / European Jou H, CYH and MEH in covery (%) a ethod I CYH 1005 980.36 9882 990.13 1014 9761 9969±0.481 the amplitude d from the div ternary mixtu igure 4, and the pyridoxine hydroc de (‐‐‐‐) and me 10 µg/mL of pyri of 20 µg/mL pyri etermination of cy de (….) in zero btracted division sp ption spectra i and 230‐243 nm of 4‐22 µg/mL e of the absor 8 nm (λ1‐λ2) and e of 3‐20 µg/m es of CYH and nge (Figure 4) obtained by app ation (4) and ed standard an ing to the follo nm (λ1– λ2) nm (λ3– λ4) ns of CYH and M re the absorptiv urnal of Chemistr synthetic mixture MEYH 101.15 98.86 97.88 99.40 100.78 10.37 99.40±1.276 value vision ure by en the hloride eclizine idoxine idoxine yclizine order pectra. in the m (λ3‐ L was rption d 215‐ L was d MEH ). The plying d (5). nd the owing (4) (5) MEH in vity (Y valu 1.63 resp solu resp app pre mea resp cou the dete 3.3. met spe to‐n of t Line rep PYH curv and CYH the bee app diff per Tab met Y1 = Y2 = Y3 = whe wav and is c and 3.4. bee tabl obt reco The dete form ry 7 (1) (2016) 3 s using the propos M PYEH 9901.38 988.18 987.50 979.30 1000.62 9900.30 999.55±1.372 ue) of CYH at ( 324 are absorp pectively. A1 a utions at the pectively. The proposed plied for determ pared mixture an percentage pectively. Table uld be successf other which ermination of t . Mean centerin thod (MCR) (m This method ectra, it eliminat noise ratio is en the method was earity of the p resented in th H, CYH and M ves relating th d 270.8 nm to H and MEH, res regression eq en obtained. Spe plication on ferent ratios o rcentage recove ble 1. The follow thod were calcu = 130.95 C1 + 10 = 6.0460 C2 + 0. = 14.394 C3 ‐ 1.4 ere Y1, Y2 and velengths, C1, C d r1, r2 and r3 ar confirmed by t d the low value . Application to After methods en applied for d lets and for PY ained results w overies and th e results obtain ermination of P mulations and 30‐36 sed spectrophotom ethod II CYYH 109.40 998.83 108.27 987.82 1000.90 989.89 109.19±1.034 (λ1‐λ2) and (λ3‐ ptivity (Y value) nd A2 are the e wavelength area under cu mination of CYH es containing d recoveries 99 e 1 is confirmin fully determine indicated that the studied drug ng of ratio spe method II) depends on t tes the derivati nhanced [15]. T s illustrated by proposed meth e range of 5‐4 MEH, respective e mean center the correspon pectively have quation parame ecificity of the m different syn of the three eries with law wing regressio ulated: 09.02, r1 = 0.999 3475, r2 = 0.999 4296, r3 = 0.999 Y3 are the pea C2 and C3 are re the correlati he high value of intercept, Ta o pharmaceuti s development determination YH and MEH were represent ey were in the ned proved that PYH, CYH and there were no metric methods. YH 01.06 9.82 00.88 8.72 00.68 8.88 00.00±0.938 ‐λ4), respective ) of MEH at (λ1‐ area under cu range (λ1‐λ2) urve method w H and PYH in th different ratios 9.40±1.276 and ng that each of ed without inte the method i gs. ctra spectroph the mean cent ive steps and th The mathematic Afkhami and B hod was prove 40, 1 -2 2 and 2 ely (Figure 5‐ ed values at 22 nding concentr been construct eters found in method has bee thetic mixtur studied drugs SD% values w n equations for 98 at 228.8 nm 99 at 262.0 nm 99 at 270.8 nm f ak amplitudes the concentrat on coefficients. of the correlat able 2. ical formulatio t and optimiza of PYH and CY in Dizerest B6 ted in the form e acceptable lim t the methods a MEH in their p o interference f 33 MEH 101.34 102.89 97.68 100.04 101.53 102.04 100.92±1.666 ely. 1.0802 and ‐λ2) and (λ3‐λ4), urve of sample ) and (λ3‐λ4), was successfully heir laboratory of them with d 99.55±1.372, the cited drugs erference from is selective for hotometric tering of ratio herefore signal‐ cal explanation Bahram [16,17] ed and it was ‐20 µg/mL for 7). Calibration 28.2, 262.0 nm rations of PYH, ted from which Table 2 have en validated by es containing s where good were obtained, r the proposed for PYH (6) for CYH (7) for MEH (8) at the selected tions in µg/mL . Good linearity tion coefficient ons ation, they had YH in Emetrex® 6® tablets. The m of percentage mit (90‐110%). are suitable for pharmaceutical from additives; d , e , y y h , s m r o ‐ n ] s r n m , h e y g d , d d L y t d ® e e . r l ; 34 Table 3. Also confirmed th Figure 5. The range of 5‐40 µ Figure 6. The range of 1‐22 µ Figure 7. The range of 2‐20 µ 3.5. Statistic Results proposed m obtained by for PYH and difference be Table 4. 3.6. Method o, results of sta he accuracy of th mean centered fir µg/mL using 0.05 N mean centered fir µg/mL using 0.05 N mean centered fir µg/mL using 0.05 N cal analysis of analysis of methods were reported HPLC d MYH [6]. Th etween them u Validation H andard addition he methods. rst ratio absorptio N as a solvent. rst ratio absorptio N as a solvent. rst ratio absorptio N HCl as a solvent. f the studied compared stat C methods for P he methods sh using student's Habib et al. / Eu n technique; Ta on spectra of PYH on spectra of CYH on spectra of MEH components b tistically with PYH and CYH [3 owed no signi ‐t and F‐ ratio ropean Journal o able 3, H in the H in the H in the by the those 3] and ificant tests, Inte [18 3.6. ana PYH 5‐4 resp PYH eva corr Tab 3.6. thei of p ran corr reco app thre in T 3.6. inte ana in t wer CYH rep con pre pre stan 3.6. to d and in T dete 3.6. and PYH line 3.3 stan corr the 3.7. resu HPL one the accu of Chemistry 7 (1 Method valid ernational Conf ]. .1. Linearity The linearity alyzing differen H, CYH and MEH 0, 4‐22 and pectively ( for m H, CYH and aluation param relation coeffic ble 2. .2. Accuracy The accuracy ir application f pure samples ges. The co responding re overies were c plication of stan ee different lev Table 3 proved a .3. Precision It was studie ermediate prec alysis of three d triplicates at th re 10, 15 and 2 H) and 4, 8 and eated using the nsecutive days cision which cise. Good re ndard deviation .4. Specificity Specificity of t different mixtu d MYH. Satisfac Table 1, confirm ermined succes .5. Limits of de d LOQ) In order to de H, CYH and ME ear range of the × N/B and LO ndard deviation responding cal high sensitivity . Statistical ana Table 2 show ults obtained b LC method [3,6 The calculated es indicating th proposed met uracy and preci ) (2016) 30‐36 dation was ference on Har of the develop nt concentratio H in triplicates. 3‐20 µg/mL method I) and 5 MEH, respecti meters like s cients were cal of these propo for determinatio of the studied ncentrations egression equa calculated as g ndard addition vels to access m accuracy of the ed with respec cision. Repeatab different concen he same day [1 20 µg/mL (for P d 12 µg/mL (fo e same concen in order to showed that esults and acc n % RSD was ob the methods wa ures containing tory results we ming that each ssfully without etection and lim etermine detect EH concentrati e calibration cu OQ = 10 × N/B n of the respon ibration curve y of the method alysis ws results of s by the propose ]. d t‐ and F‐value at there is no s hods and the r ision. carried out rmonization (I ped methods w ons of standar . It was evaluat for PYH, CY 5‐40, 1‐22 and 2 ively (for me slopes, interce culated and ar sed methods w on of different drugs within were obtaine ations then th given in Table technique was method accuracy e proposed meth ct to both rep bility was calcu ntrations of pu 19]. The used PYH), 4, 8 and or MEH). The ex trations three determine the the developed ceptable perce btained, Table 2 as proved by th different ratio ere obtained an h of the cited d interference fro mits of quantita tion and quant ons in the low urve and the eq B were used, w nse and B is th [20]. The low v ds, Table 2. statistical comp ed methods and es are less than significant diffe reported ones w according to CH) guidelines was proved by rd solutions of ed in the range YH and MEH, 2‐20 µg/mL for ethod II). The epts and the re presented in was checked by concentrations their linearity ed from the he percentage 2. In addition, carried out on y. Results given hods. peatability and ulated through re components concentrations 10 µg/mL (for xperiment was times on three e intermediate d methods are entage relative 2. heir application os of PYH, CYH nd represented drugs could be om the other. ation (LOD ification limits, wer part of the quations LOD = where N is the he slope of the values indicate parison of the d the reported the theoretical erence between with respect to o s y f e , r e e n y s y e e , n n d h s s r s e e e e n H d e , e = e e e e d l n o Habib et al. / European Journal of Chemistry 7 (1) (2016) 30‐36 35 Table 2. Regression and analytical parameters of the proposed methods for determination of PYH, CYH and MEH. Method II Method I Parameters MEHCYH PYHMEHCYHPYH 2‐201‐22 5‐403‐204‐225‐40Calibration range (µg/mL) 14.394 6.0461 130.59 ‐ ‐ ‐ Slope ‐1.42960.3475 109.02‐‐‐Intercept 0.99990.9999 0.9998‐‐‐Correlation coefficient 99.98100.08 99.8899.97100.0599.76Accuracy 1.151.05 0.511.160.290.69Repeatability (%RSD)a 1.401.67 0.711.300.890.85Intermediate precision (%RSD)b 0.530.23 1.100.801.101.06LOD c 1.60 0.70 3.00 2.40 3.30 3.20 LOQ d a The intra‐day precision (n = 9), average of three different concentrations repeated three times within day. b The intr‐day precision (n = 9), average of three different concentrations repeated three times in three successive days. c LOD = (SD of the response /slope) × 3.3. d LOQ = (SD of the response /slope) × 10. Table 3. Determination of studied drugs in Emetrex® and Dizerest B6® tablets by the proposed spectrophotometric methods and the application of standard addition technique. Standard additionFound (%±SD) a Taken (µg/mL) ComponentPharmaceutical formulation Method Found (%)Found (µg/mL) b Added (µg/mL) 101.006.06 6.0097.56±1.530 6.00 PYH Emetrex® tablets claimed to contain 30 mg of PYH and 50 mg CYH (Batch No. 20766) I 101.6010.16 10.00 99.0714.86 15.00 Mean±SD = 100.56±1.079 99.253.97 4.00105.24±0.080 10.00 CYH 100.0010.00 10.00 102.0012.24 12.00 Mean±SD = 100.42±1.161 100.805.04 5.001.642±104.3210.00 PYH Dizerest B6® tablets claimed to contain 50 mg of PYH and 25 mg MEH (Batch No. 30248) I 98.209.82 10.00 97.5314.63 15.00 Mean±SD = 98.84±1.411 100.673.02 3.001.624±101.985.00 MEH 99.004.95 5.00 98.009.80 10.00 Mean±SD = 99.22±1.014 99.175.59 6.001.460±99.85 6.00 PYH Emetrex® tablets claimed to contain 30 mg of PYH and 50 mg CYH (Batch No. 20766) II 100.3010.03 10.00 98.7314.81 15.00 Mean±SD = 99.40±0.661 101.673.05 3.00103.73±0.73110.00 CYH 101.2110.12 10.00 100.7512.09 12.00 Mean±SD = 101.21±0.376 102.405.12 5.001.464±104.83 10.00 PYH Dizerest B6®. tablets claimed to contain 50 mg of PYH and 25 mg MEH (Batch No. 30248) II 101.7010.17 10.00 99.3314.90 15.00 Mean±SD = 101.14±1.314 101.003.03 3.001.126+100.10 5.00 MEH 99.404.97 5.00 97.109.71 10.00 Mean±SD = 99.17±1.600 a Average of six determination. b Average of three determination. Table 4. Statistical comparison between the results obtained by the proposed spectrophotometric methods and the reported methods for the determination of pyridoxine hydrochloride, cyclizine hydrochloride and meclizine hydrochloride in pure powder form. Reported methods Method IIMethod I Component MEH b CYH a PYH a MEH CYH PYH MEH CYH PYH 100.06100.11 99.0099.98100.0999.8899.97100.05 99.76 Mean 0.8191.137 1.3060.9690.9751.1220.9991.199 1.663 SD 0.6711.293 1.7050.9390.9511.2590.9981.438 2.766 Variance 77 77777 7 7 N ‐‐ ‐0.136 0.0836 1.252 0.152 0.085 0.887 t‐Test (2.179) c ‐‐ ‐1.401 1.360 1.355 1.489 1.114 1.622 F‐Test (4.284) c a HPLC determination of PYH and CYH using acetonitrile/0.05 M KH2PO4 (50:50, v:v, pH = 4.0) as developing system and detection at 239 nm [3]. b HPLC determination of PYH and MEH using phosphate buffer / acetonitrile / trifluoroacetic acid (30:70:0.1, v:v:v) as developing system and detection at 254 nm [6]. c The values between parenthesis are corresponding to the theoretical values of t and F (p = 0.05). 4. Conclusion Selective spectrophotometric methods have been established for determination of PYH, CYH and MEH in their ternary mixture for the first time. The developed methods are considered to be simple, selective and could be used for determination of the cited drugs (PYH, CYH and MEH) either in their laboratory prepared mixtures or in their pharmaceutical formulations (Emetrex® tablets and Dizerest B6® tablets). The validity of the methods has been ascertained by the good results which obtained by applying the standard addition techniques confirming that it is valuable for application in quality control laboratories for determination of the studied drugs. 36 Habib et al. / European Journal of Chemistry 7 (1) (2016) 30‐36 Acknowledgement The authors would like to express their appreciation and thanks to AMOUN Pharmaceutical Company and SIGMA Pharmaceutical Company for the provision of the necessary materials to carry out this work. References [1]. Budavari, S. The Merck Index, an encyclopedia of chemicals, drugs and biological, 13th Ed. Merck and Co., Inc. White House Station, NJ, USA, 2002. [2]. 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