untitled ISSN 215 Simulta HCl in th densitom Maha Moh Nouruddi Pharmaceutical * Corresponding Tel.: +2.010.440 ARTICLE IN DOI: 10.5155/e Received: 12 No Received in rev Accepted: 16 De Published onlin Printed: 31 Mar KEYWORDS Validation Methyl paraben Pseudoephedri HPTLC‐densito Carbinoxamine Pharmaceutical 1. Introduct Chemica 2‐[(4‐chlorop amine (2Z)‐2 amine deriv rinic, signific used for the a common in matic treatm Chemica (1S,2S)‐2‐(m [2]. It is a ste PSH and its s of nasal con ingredients and cold sym A review maleate has and first‐ord [6,7], LC‐MS amine in hu 53‐2249 (Print) neous de heir pure metric me hamed Abde n Wageih Al l Analytical Chemis g author at: Pharm 08208. Fax: +2.082.2 FORMATION eurjchem.7.1.37‐41 ovember 2015 vised form: 02 Dece ecember 2015 ne: 31 March 2016 rch 2016 S n ne HCl metry e maleate l formulation tion lly, carbinoxam phenyl)(2‐pyrid 2‐butenedioate vative, a sedati cant sedative an relief of allergi ngredient of com ment of coughs a lly, pseudoeph methylamino)‐1 ereoisomer of e salts are given b ngestion and ar in preparation mptoms (Figure w in the litera been determin der UV‐spectros S/MS method f man plasma [8 / ISSN 2153‐225 ht Europ terminati form and ethod elrahman, Eg li and Raghd stry Department, Fa aceutical Analytica 2317950. E‐mail ad 1.1355 ember 2015 mine maleate (C dinyl)methoxy] (Figure 1) [1]. ing antihistami nd serotonin an ic conditions su mpound prepar and the commo hedrine HCl (PS ‐phenylpropan ephedrine that by mouth for th re commonly c ns intended for 1) [1]. ature revealed ned by spectrop scopic method for the quantif 8], and gas liqu European Journ Europ 57 (Online)  20 ttp://dx.doi.org pean Jo Journal web ion of car d in their glal Abdelha da Abdelmo aculty of Pharmacy al Chemistry Depar ddress: raghdaema ABSTRACT An accurate, s method has b pseudoephed separation wa methanol:ethy were scanned its linearity w and pseudoe determination interference comparison o the reported method is sen the studied dr Cite this: Eur. RM) is designa ]‐N,N‐dimethyle . It is a monoet ine with antim ntagonist effect uch as rhinitis, rations for symp n cold (Figure 1 SH) is designat ‐1‐ol hydroch has the same a he symptomatic combined with r the relief of that carbinoxa photometric me [3‐6], HPLC m fication of carb uid chromatogr al of Chemistry 7 pean Journal of C 016 Atlanta Pub /10.5155/eurjch ournal bpage: www. rbinoxami pharmace ameed Abde neim Emam y, Beni‐ Suef Univer rtment, Faculty of P am88@gmail.com ( sensitive and se been developed rine HCl in their as performed on ylacetate:triethy d at 215 nm. The was evident in th ephedrine HCl, n of the above m from methyl p of the results ob HPLC method s nsitive, accurate rugs. . J. Chem. 2016, ted as ethan thanol musca‐ ts. It is and is ptom‐ 1) [1]. ted as loride action. c relief other cough amine ethods method binox‐ raphic met com spe pho stab 7 (1) (2016) 37‐ Chemistry lishing House LL hem.7.1.37‐41.1 of Che .eurjchem.co ine malea eutical fo elaleem, m * rsity, 62514, Beni‐S Pharmacy, Beni‐ Su (R.A. Emam). elective High Per and validated f r binary mixture n aluminum plat ylamine (5:4:2:0 e proposed meth e ranges of 0.4‐8 respectively. T mentioned drug paraben presen btained by the d showed no sign and precise and 7(1), 37‐41 thod [9], while mponents in dif ectrophotometr otodiode array bility indicating N OH H N Figure 1. Chem ‐41 LC ‐ All rights re 1355 emistry m ate and ps rmulation Suef, Egypt uef University, 6251 rformance Thin for determinatio e without previo tes precoated wi 0.2, v:v:v:v) as a hod has been va 8.0 and 4‐24 µg/ The developed gs in their pharm nt as excipient developed HPTL ificant differenc d can be easily u for PSH, it has fferent pharma ic methods [1 spectroscopy g HPLC method O N Cl H N H Cl (b) mical structure of C served ‐ Printed y seudoeph n by HPTL 4, Beni‐Suef, Egypt Layer Chromat on of carbinoxam ous separation. ith silica gel 60F a developing sy alidated as per IC / band for carbin method has b maceutical form t has been det C method and t ce between them sed for quality c s been determin ceutical formul 0,11] and sec [12], HPLC me [18]. O HO O OH HO CRM (a), PSH (b) a d in the USA hedrine LC‐ t. tography (HPTLC mine maleate an Chromatograph F254 using aceton ystem. The band CH guidelines an noxamine malea been applied fo mulation where n tected. Statistic those obtained b m. The develope control analysis ned with other lations such as, ond derivative ethods [13‐17], (a) O O (c) and MP (c). C) nd hic ne: ds nd ate or no cal by ed of r , e , 38 Abdelrahman et al. / European Journal of Chemistry 7 (1) (2016) 37‐41 The only reported method for determination of both drugs simultaneously was HPLC method [19] without taking in consideration presence of methyl paraben as formulation excipient. Methyl paraben (MP) is a hydroxybenzoates preservative; alkyl esters of p‐hydroxy benzoic acid, with antibacterial and antifungal properties (Figure 1). Hydroxy benzoates are used as preservatives in pharmaceutical preparations in usual concentrations of up to 0.25% [1]. The advantage of HPTLC is that several samples can be run simultaneously using a small quantity of developing system, unlike HPLC, thus lower in analysis time and cost per analysis. So, the focus of the present study was to develop an accurate, specific and selective method for simultaneous determination of carbinoxamine maleate and pseudoephedrine HCl without interference from oral drops excipient (methyl paraben). 2. Experimental 2.1. Instrumentation A sample applicator for TLC method was linomat V applicator with 100 µL syringe, (Camage, Muttenz, Switzerland). TLC scanner, densitometer, (Camag, Switze‐ rland) was used controlled by WINCATS software, (V 3.15, Camage). HPTLC aluminum plates (20×20 cm) coated with 0.25 mm silica gel 60F254, (Merck, Germany), were used. UV lamp with short wavelength 254 nm, (VL‐6.LC, MARNE LA VALLEE cedex 1, FRANCE ), was used. Also, sonix TV ss‐series ultrasonicator (USA) was used. There are some parameters that had been adjusted during TLC scanning; radiation source was a deuterium lamp, Slit dimension was 6×0.45 mm, scanning mode was "absorbance" and scanning speed was 20 mm/s. 2.2. Materials and reagents 2.2.1. Pure standards Carbinoxamine maleate and pseudoephedrine HCl pure standards were kindly supplied by Medical Union Pharmaceuticals, Abu‐Sultan, Ismailia, Egypt with purity of 99.69 and 98.83%, respectively, according to the manufacturer certificate. Methyl paraben was kindly supplied by the Pharaonia Pharmaceuticals, Alexandria, Egypt, with purity of 99.02% according to the manufacturer certificate. 2.2.2. Pharmaceutical formulation Rhinostop® oral drops (batch No. 134089) was Labeled to contain 2 mg CRM and 25 mg PSH in each mL, was manufactured by Medical Union Pharmaceuticals, Abu‐Sultan, Ismailia, Egypt. 2.2.3. Chemicals and solvents All chemicals used throughout this work were of analytical grade and were used without further purification: Methanol, acetone, ethylacetate, triethylamine, (El‐Nasr Pharmaceutical Chemicals Co., Abu Zabaal, Cairo, Egypt). 2.2.4. Solutions Stock standard solutions of CRM and PSH (1 and 4 mg/mL, respectively): 100 and 400 mg of CRM and PSH, respectively, were accurately and separately weighed into two 100 mL volumetric flasks and dissolved in methanol. Working standard solutions of CRM and PSH (0.1 and 0.2 mg/mL): They were prepared by diluting 10 and 5 mL each of CRM and PSH, respectively, from their respective stock solutions into two separate 100 mL volumetric flasks and the volume was completed to the mark with methanol. Preparation of pharmaceutical formulation: 4 mL of Rhinostop® oral drops equivalent to 100 mg PSH and 8 mg CRM were accurately transferred into a 100 mL volumetric flask and then the volume was completed to the mark with methanol to prepare 1 mg/mL PSH (and the corresponding amount of 80 µg/band CRM) stock solution from which a working solution of 0.1 mg/mL PSH (corresponding to 8 µg/band of CRM) was prepared in methanol. 2.3. Procedure 2.3.1. Chromatographic conditions It was performed using pre‐coated silica gel HPTLC aluminum plates (20×10 cm). The plates were pre‐washed with methanol and activated at 100 °C for 15 minutes prior to samples application. Samples were applied in the form of bands (6 mm length, 5 mm spacing and 10 mm from the bottom edge of the plate). Linear ascending development was performed in a chromatographic tank previously saturated with acetone: methanol:ethylacetate:triethylamine (5:4:2:0.2, v:v:v:v) for half an hour at room temperature to a distance of about 80 mm. The developed plates were air dried and then scanned at 215 nm. 2.3.2. Linearity and construction of calibration curves Aliquots equivalent to 0.4‐8.0 and 4‐24 mg were accurately and separately transferred from CRM and PSH stock standard solutions (1 and 4 mg/mL), respectively, into a series of 10 mL volumetric flasks and the volume was completed to the mark with methanol, 10 µL of each solution were applied in triplicates on the pre‐washed HPTLC plates to obtain the concentration range of 0.4‐8.0 and 4‐24 µg/band for CRM and PSH, respectively. The procedure under chromatographic conditions was then followed. The integrated peak area was then recorded and a calibration curve for each drug was constructed by plotting the integrated peak area versus the corresponding concentration of each drug and the regression equations were then computed. 2.3.3. Application to pharmaceutical formulation The procedure under linearity was followed on Rhinostop® oral drops and the concentrations of CRM and PSH were determined using the computed regression equations. To assess the accuracy of the proposed method, standard addition technique was applied. 3. Results and discussion The main task of this work was to develop sensitive, selective and accurate HPTLC‐densitometric method for the determination of CRM and PSH in their binary mixture and in Rhinostop® oral drops without interference of Rhinostop® excipient; methylparaben with satisfactory precision for good analytical practice (GAP). Thin layer chromatography has become a well‐established technique for the assay of drugs either in binary or in multi‐ component mixtures [20‐27]. The proposed method is based on the difference in the retardation factor (RF) between the CRM, PSH and MP. 3.1. Method optimization The experimental conditions had been optimized to get the desired chromatographic resolution and efficiency with sharp symmetric peaks. 3.1.1. Developing system Table 1. Regres Parameters Linearity Range (µg/ban Slope Intercept r (correlation c Accuracy Precision (%R Repeatability a Intermediate p Roubustness ( Saturation tim Triethylamine Scanning wave LOD* (µg/band LOQ* (µg/band a The intraday ( b The interday ( * LOD and LOQ Table 2. Determ application of s Pharmaceutic Rhinostop® ora Labeled to cont Initially, select a suita of the three s tried to p chloroform: acetone:meth methanol:eth the first sy separation a chloroform b value of PS Addition of and MP pea PSH. Additio PSH peak s between the 3.1.2. Satura The sat important to minimizing t during the de system has b 3.1.3. Scann Different nm) were t method; scan considerable While scanni CRM which them. Rf valu and 0.77 for a developin acetate:trieth densitogram separated. ssion parameters o nd) coefficient) RSD) a precision b ( % RSD) me ±5 min ratio ±0.02 mL elength ±1 nm d) d) (n = 3), average of t (n = 3), average of values were calcu mination of carbin tandard addition t cal formulations al drops (B.N. 1340 tain 2 mg CRM and trial experime able developing studied compon ermit good methanol (4:6 hanol:ethyl ac hyl acetate:triet ystem, tailed and very low Rf by acetone in t SH but gave t ethyl acetate in aks shape and on of TEA (0.2 shape and sym three compone ation time uration time o achieve hom the evaporation evelopment. Th been optimized ing wavelengt t scanning wav tried in order nning at 254 n e sensitivity for ing at 215 nm g is very import ues of the sepa PSH, CRM and ng system c hyl amine (5:4 m Figure 2, the Abdelrahman of the proposed HP three different con three different con lated using a visua oxamine maleate a technique. 089) d 25 mg PSH/mL ents were perf g system for the nents. Several s separation wi 6, v:v), acetone cetate (5:4:2, thyl amine (5:4 peaks were f value of PSH p the second syst ailed peaks w n the third sys symmetry but mL) to the thir mmetry with t ents. required befo mogeneity of th n of the solven he saturation tim and found to b th velengths (210 to enhance th m gave bad sen r CRM and hig gave the best se tant for quality arated compone MP, respective containing ace 4:2:0.2, v:v:v:v) e peaks are s et al. / European PTLC‐Densitometr ncentrations repea ncentrations repea al non‐instrumenta and pseudoephedr Tak (µg CRM 1 PSH 12. formed in a vi e suitable sepa solvent systems ithout tailing, :methanol (6:4 v:v:v) and ace 4:2:0.2, v:v:v:v). obtained with peak. Replacem tem increased with bad separ stem, improved gave broad pe rd system, imp the best separ ore developme he atmosphere, nt from HPTLC me of the devel e 30 min. 0, 215, 220 and he sensitivity o nsitivity for PS h sensitivity fo ensitivity for PS y control analy ents were 0.33 ly, obtained by etone:methanol ). As shown i symmetric and n Journal of Chem ric methods for det ated three times wi ated three times in al method [24]. rine HCl in pharma ken g/band) % Rec 95.74 5 106.6 ew to ration s were e.g., 4, v:v), etone: Using h bad ment of the Rf ration. d CRM eak of proved ration ent is thus, plate loping d 254 of the H and or MP. SH and ysis of 3, 0.46 y using l:ethyl in the d well Figu carb acet deve and μg/ the A1 = A2 = whe con coe par and Rhi app ther mistry 7 (1) (201 termination of carb C 0 1 9 0 9 0 1 0 0 0 0 0 ithin day. n three successive d aceutical formulati covery±SD St P 4±1.489 0 1 1 M 64±1.442 4 6 8 M ure 2. HPTLC den binoxamine malea tone:methanol:ethy eloping system. A linear relati d PSH and the i /band and 4‐24 regression equ = 1880.40×C1+ = 213.46×C2 + 2 ere A1, A2 are ncentrations in fficients of CRM rameters are giv The validity o d PSH was stu nostop® oral d plying standard re was no inter 6) 37‐41 binoxamine maleat RM .4‐8.0 880.40 43.54 .9999 9.51 .981 .608 .147 .603 .073 .12 .36 days. on by the propose tandard addition Pure added (µg/ba .8 .0 .2 Mean±SD .0 .0 .0 Mean±SD sitogram of (a) ps te (Rf = 0.46), (c) ylacetate:triethyla ionship betwee ntegrated peak μg/band for CR uations were co 943.54 r1 = 0.99 290.64 r2 = 0.99 e the integrate n μg/band and M and PSH, resp ven in Table 1. f the proposed died by applyi drops, Table 2 d addition tec rference from M te and pseudoephe PSH 4‐24 213.46 290.64 0.9998 99.90 1.180 1.432 0.551 0.788 0.864 1.16 3.50 d HPTLC‐Densitom n technique and) % Rec 99.89 98.12 100.84 99.62± 100.74 98.79 101.75 100.43 seudoephedrine H ) methylparaben mine (5:4:2:0.2, en the concentr ks area in the r RM and PSH, re mputed and fou 999 for CRM 998 for PSH ed peak area, d r1, r2 are t pectively. Regre d method for an ing the propos . It was furthe chnique, which MP, Table 2. 39 edrine HCl. 6 4 8 metric method and covery 4 ±1.380 4 5 3±1.505 HCl (Rf = 0.33), (b) (Rf = 0.77) using v:v:v:v) as a rations of CRM range of 0.4‐8.0 espectively, and und to be: (1) (2) C1, C2 are the the correlation ession equation nalysis of CRM sed method on er assessed by h showed that d ) g a M 0 d e n n M n y t 40 Abdelrahman et al. / European Journal of Chemistry 7 (1) (2016) 37‐41 Table 3. Statistical comparison of the proposed method and the reported one for determination of CRM and PSH in their pharmaceutical formulation. Items HPTLC Reported method HPLC [19] b CRM PSH CRM PSH Mean 99.51 99.90 99.97 101.06 SD 1.584 1.004 1.2895 0.854 RSD% 1.592 1.005 1.2899 0.845 n 8 8 6 6 Student's t‐test 0.576 (2.178) a 1.748 (2.178) a F‐value (3.972) 1.508 (4.875) a 1.384 (4.875) a a Figures between parenthesis represent the corresponding tabulated values of t and F at p= 0.05. b An isocratic reversed phase LC analysis conducted on a CN column, with a mobile phase consisting of acetonitrile‐methanol‐phosphate buffer (pH = 5.3)‐water (140:170:40:100) and at a detection wavelength of 262 nm and a flow rate of 1 mL/min. Statistical analysis of the results obtained by applying the developed HPTLC‐densitometric method with those obtained by the reported HPLC method [19], showed no significant difference with 95% confidence regarding both accuracy and precision, Table 3. 3.2. Method validation Method validation was performed according to ICH guidelines [28]. 3.2.1. Linearity and range Linearity of the proposed method was evaluated and it was evident in the range of 0.4‐8.0 and 4‐24 µg/band for CRM and PSH, respectively. Good linearity was evident from the high value of correlation coefficient as shown in Table 1. 3.2.2. Accuracy Accuracy of the method was checked by applying the proposed method for determination of different blind samples of pure CRM and PSH. The concentrations were calculated from the corresponding regression equations and the results were presented in Table 1. Accuracy of the method was further assured by applying the standard addition technique on pharmaceutical formulation where good recoveries were obtained revealing no interference from excipients, as given in Table 2. 3.2.3. Precision Repeatability: Three concentrations (0.8, 1.0 and 2.0 µg/band) of CRM and (8, 10 and 14 µg/band) of PSH were analyzed three times intra‐day using the proposed method. Good %RSD values were obtained confirming the repeatability of the method as given in Table 1. Intermediate precision: The previous procedure was repeated inter‐day on three different days for the analysis of the three chosen concentrations. Acceptable % RSD values were obtained as given in Table 1. 3.2.4. Specificity The specificity of the proposed method was demonstrated by the HPTLC chromatogram as shown in Figure 2 where complete separation of CRM, PSH and MP was achieved. Also, the absence of any peaks at the retention time of the studied drug and the good results obtained on applying the method to Rhinostop® oral drops, presented in Table 2, prove that there was no interference from excipients. 3.2.5. Limit of detection and limit of quantitation ICH recommendations [24] were followed using a visual non‐instrumental method to calculate the values of LOD and LOQ of the two studied components where LOD is the concentration at which the signal to noise ratio is equal to 3:1 while LOQ is the concentration at which the signal to noise ratio is equal to 10:1. Low values of both LOD and LOQ indicated the high sensitivity of the developed method, Table 1. 3.2.6. Robustness Robustness is the capacity of the method to remain unchanged with small deliberate variations in method parameters e.g. changing triethyl amine volume in the developing system 2±0.1 mL, changing saturation time 30±5 min and changing the scanning wavelength ±3 nm. The low value of %RSD shows that the method is robust and that the deliberate small change in the studied factors did not lead to significant changes in Rf values, area or symmetry of the peaks. The % RSD was calculated, and the results are given in Table 1. 3.2.7. System suitability An overall system suitability testing was done to determine if the operating system were performed properly. Parameters including resolution (Rs), selectivity factor (α) and peak asymmetry (tailing factor) were calculated where good results were obtained as given in Table 4. Table 4. System suitability testing parameters of HPTLC densitometric method. Parameters CRM PSH Reference values [29] Resolution (Rs) 2.55 > 1.5 Selectivity factor (α) 1.39 >1 Tailing factor (T) 1.01 0.9 ‐ The results obtained by applying the proposed method were statistically compared with those obtained by applying the reported HPLC method [19] and the calculated F and t‐ values showed no significant differences between them, as shown in Table 3. 4. 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