untitled ISSN 215 Develop the simu degrada Mohamed Ehab Faro 1 Medicinal Chem 2 Pharmaceutica 3 Central Admin * Corresponding Tel.: +2.012.243 ARTICLE IN DOI: 10.5155/e Received: 06 No Received in rev Accepted: 29 No Published onlin Printed: 31 Mar KEYWORDS RP‐LC Otilonium brom p‐Aminobenzoi Stability indicat Pharmaceutical Simultaneous d 1. Introduct Otiloniu (octyloxy)ben (Figure 1). derivatives w hypersensiti particular fo [1]. Irritable the wide ran the numerou the main re etiologic the muscle of th having a loca Furthermore rapidly elimi to be well t effects that muscarinic s 53‐2249 (Print) pment and ultaneous ation prod d Mahmoud ouk Elkady 2 mistry Department al Chemistry Depar istration for Pharm g author at: Centra 314304. Fax: +2.012 FORMATION eurjchem.7.1.97‐10 ovember 2015 vised form: 28 Nov ovember 2015 ne: 31 March 2016 rch 2016 S mide ic acid ting assay l preparation determination tion m bromide is nzoyl]amino}be OB is a memb widely used for vity disorders r the treatment e bowel syndro nge of symptom us pathological eason of the l erapy [2,3]. OB e colon and rec al action with n e, OB does not c inated from ma tolerated, and are typical spasmolytic age E / ISSN 2153‐225 ht Europ d validati s determi duct in bu El‐Kerdawy 2 and Ahmed t, Faculty of Pharm rtment, Faculty of P maceutical Affairs, M al Administration fo 2.24314304. E‐mai 01.1358 ember 2015 N, N‐diethyl‐ enzoyl)oxy]etha ber of the qua the treatment of the intesti t of Irritable Bo ome is a compl ms cannot be a mechanisms h acking of an B shows specifi ctum at therape negligible system cross the blood ajor organs [7]. it seems to be of the system ents [5,8]. European Journa Europ 57 (Online)  20 ttp://dx.doi.org/ pean Jo Journal web on of a st nation of ulk drug a y 1, Ramzia I d Adel Othm acy, Mansoura Uni Pharmacy, Cairo Un Manyal, Cairo 1156 or Pharmaceutical A l address: a.osman8 ABSTRACT A simple, pre been develop expected de pharmaceutic on Waters Atl (pH = 2.35) co was 0.8 mL/m range of 0.5‐1 of 1‐50 μg/m were found to respectively. applied to the Cite this: Eur. N‐methyl‐2‐[(4 anaminium bro aternary ammo of hypermotilit ine, and is us owel Syndrome ex disease, in w always attribut hypothesized. T appropriate sp icity for the sm eutic concentra mic absorption d‐brain barrier . OB has been s e devoid of the mically acting al of Chemistry 7 pean Journal of C 016 Atlanta Pub /10.5155/eurjch ournal bpage: www. tability‐in f otilonium and pharm smail El‐Bag man 3,* iversity, Mansoura 3 niversity, Cairo 115 62, Egypt Affairs, Manyal, Ca 88@yahoo.com (A. cise, accurate an ped for the sim gradation prod cal preparation. lantis C18 colum ontaining 0.05% min with UV det 100 μg/mL with mL with regressi o be 0.0665 and The method wa e determination . J. Chem. 2016, 4‐{[2‐ omide onium ty and sed in e (IBS) which ted to That is pecific mooth ations, [4‐6]. and is shown e side anti‐ for biol [6,1 mix [13 Figu 7 (1) (2016) 97‐1 Chemistry lishing House LL hem.7.1.97‐101.1 of Che .eurjchem.co ndicating R m bromid maceutica gary 2, 35516, Egypt 562, Egypt airo 11562, Egypt. A. Othman). nd rapid stabilit multaneous dete duct; p‐aminob Chromatograph mn (4.6 × 150 m % TFA: acetonitri tection at 290 n regression coef ion coefficient o d 0.2018 μg/mL as validated as p of the drug in bu 7(1), 97‐101 There are few analysis of oti logical fluids in 11] and LC‐MS/ xtures with be ] and spectroph ure 1. Chemical str 101 LC ‐ All rights re 1358 emistry m RP‐LC me de and its al prepara ty indicating liqu ermination of o benzoic acid ( ic separation wa mm, 5 µm) using ile (30:70, v:v) a nm. Linearity wa fficient of 1 for O of 0.9998 for PA L for OB and 0.1 per ICH guidelin ulk powder and w analytical me ilonium bromid ncluding LC wit /MS [12]. Besid enzodiazepines hotometric met O N H O O H2N ructures of OB (a) served ‐ Printed y ethod for expected ation uid chromatogra otilonium brom (PABA) in bu as carried out by g 2 mM ammoniu as the mobile ph as obtained ove OB, and over con ABA. The values 1974 and 0.598 nes. The method pharmaceutical ethods reported de either in do th UV detection des, it has been by capillary e thods [14‐17]. O O N+ Br- ( O OH (b) and its degraded p d in the USA d aphy method ha ide (OB) and i ulk powder an y isocratic elutio um acetate buff ase. The flow ra er a concentratio ncentration rang of LOD and LO μg/mL for PAB d was successful preparation. d in literatures osage forms or n [9,10], LC‐MS determined in electrophoresis (a) product PABA (b). ad its nd on fer ate on ge OQ A, lly s r S n s 98 Otilonium readily und benzamido)b molecule [1 hydrolysis to one of the de present wor stability‐indi nation of O pharmaceuti 2. Experime 2.1. Instrum An Agile vacuum dega detector was Waters Atlan processor m vortex mixer 2.2. Reagent All chem grade. OB ( Egypt. p‐am buffer analyt Acetonitrile Scientific, UK 40 mg OB w water was U.S.A. 2.3. Chroma Chromat Atlantis C18 isocratic elut ammonium acetonitrile through the performed a detection wa 2 µL. 2.4. Prepara Accurate mL volumet volume with 2.4.1. Prepa Five mL volumetric f acetonitrile ( 2.4.2. Prepa Accurate 25 mL volum volume with 2.4.3. Prepa Five mL volumetric f acetonitrile ( 2.5. Sample Ten Sp weighted, cr m bromide de derwent hydr benzoic acid t 8], and the se o amine [19]. T egraded produc rk aimed to de icating method OB and PABA ical preparation ental mentation ent 1260 HPLC asser, quaterna s used. Separat ntis C18 column model KBK 420 r (Berlin) were ts and referenc micals and reage >99%) was ki minobenzoic aci tical grade wer (HPLC grade K. Spasmomen® was purchased produced in‐h atographic cond tographic sepa 8 column (4.6 tion based on a acetate buffer (30:70, v:v). column at a flo at controlled as carried out a ation of OB stan ely weight 25 m tric flask and d acetonitrile (1 ration of OB w of OB stock so flask; the volu (100 μg/mL). ration of PABA ely weight 25 m metric flask and acetonitrile (1 ration of PABA of PABA stock s flask; the volu (100 μg/mL). solution prepa asmomen® 4 ushed then mix El‐K egraded at roo rolysis to yi through the e econdary amid Therefore PABA cts of OB from evelop a fast, r d for the sim (Figure 1) in n. system (Germ ary‐pump, colum ion and quantit n (4.6 × 150 m 00 (Germany) a used. ce samples ents used were o indly provided id (99%) and re purchased fr e) was purch ® 40 mg tablet n from a local p house by Barn ditions aration was a × 150 mm, a mobile phase (pH = 2.35) The mobile p w rate of 0.8 m column temp t 290 nm. The i ndard stock so mg of OB was t dissolved in an mg/mL). working solutio olution was tra ume was comp A stock solution mg of PABA wa d dissolved in a mg/mL). A working solu solution was tr ume was comp aration 40 mg tablet xed well. Accur Kerdawy et al. / E m temperatur eld 4‐(2‐(octy ester group in de group unde A is predicted both hydrolysi obust and econ multaneous det n bulk powder many), equipped mn oven and U tation were ma mm, 5 µm). Ultra and SCILOGEX of analytical or d by Minapharm ammonium a om Merck, Ger hased from F nominally conta pharmacy. Deio nstead diamond achieved on W 5 µm) by app e consisting of contain 0.05% phase was pu mL/min. Analysi erature 25 °C injection volum olution transferred into nd completed t n ansferred to a 5 pleted to mark n as transferred and completed tion ansferred to a 5 pleted to mark ts were accu rately weighed European Journa e and yloxy) n the rwent to be s. The nomic termi‐ r and d with UV/VIS ade on asonic MX‐S HPLC m Co, cetate many. Fisher aining onized d RO, Waters plying 2 mM % TFA: umped is was C and me was o a 25 to the 50 mL k with into a to the 50 mL k with urately 128.1 mg bro abo son volu solu use 2.6. 2.6. tran volu solu ren into to v 50 inje incl dete obt con 2.6. usin 5‐4 2.6. PAB for pre and com con eac Figu labo 2.6. dilu trip calc Figu sam al of Chemistry 7 of the powde omide, were tra out 30 mL ac nicated for 15 m ume was com ution was filter ed as the sample . Procedure .1. Constructio Different aliq nsferred into umes were co utions containi t aliquots from o another serie volumes with m μg/mL PABA. ected in triplica luding the mo ection at 290 n ained by plot ncentration (C). .2. Assay of OB The procedure ng concentratio 5 μg/mL PABA .3. Assay of OB BA The procedure simultaneous d pared mixture d 1.2‐3.5 µg/m mpleted to the m ncentrations of h from its calib ure 2. A typical ch oratory prepared m .4. Assay of OB The sample so uted, completed plicates (Figur culated using its ure 3. A typical L ple. (1) (2016) 97‐10 er tablets equ ansferred to 50 cetonitrile wer min with inter pleted to the ed discarding fi e solution (1 mg on of calibratio quots from O a series of 10 ompleted with ng 0.5‐100 μg/ m PABA workin s of 10 mL volu mobile phase to A volume of ates into the ch obile phase at nm were adjus tting area und B and PABA in b e mentioned in ons equivalent t in bulk, respec B in laboratory e mentioned in determination o s using concen mL of OB an mark by mobile f OB and PABA ration equation hromatogram of 75 mixture. B in pharmaceu olution prepare d with mobile re 3). The c s calibration eq C chromatogram 01 uivalent to 50 mL volumetric re added. The rmediate shakin mark with ac irst 10 mL and g/mL). n curves OB working s 0 mL volumet h the mobile /mL OB. Altern ng solution we umetric flasks, o give solutions 2 µL of each hromatograph. flow rate 0.8 sted. Calibratio der the peak bulk n Section 2.6.1 to 3‐70 μg/mL tively. prepared mixt n Section 2.6.1 of OB and PABA ntrations equiv nd PABA, res e phase (Figure A were separa n. 5 µg/mL OB and 1 utical preparat ed in Section 2 phase and th oncentrations quation. of 50 µg/mL of o mg otilonium c flask to which solution was ng. Finally, the cetonitrile. The the filtrate was solution were tric flasks, the phase to give natively, differ‐ ere transferred and completed s containing 1‐ h solution was The conditions 8 mL/min and on curves were (AUP) against 1 was repeated OB in bulk and tures with 1 was repeated A in laboratory valent to 10‐96 pectively, and e 2). Recovered ately calculated 1.5 µg/mL PABA in tions 2.5 was serially hen injected in of OB were otilonium bromide m h s e e s e e e ‐ d d ‐ s s d e t d d d y 6 d d d n y n e e El‐Kerdawy et al. / European Journal of Chemistry 7 (1) (2016) 97‐101 99 Table 1. System suitability tests for the proposed LC method. Data for PABA Data for OB Item 6674 6320N, No of theoretical plates per column 1.07 1.16 T, Tailing factor 0.101 0.170%R.S.D. of six injections peak area 0.08 0.25% R.S.D of six injection of retention time (min) 14.63 14.63R, Resolution factor %R.S.D: % Relative standard deviation. Table 2. Results obtained by the proposed LC method for the determination of OB and PABA. Item OB PABA Retention time 4.97±0.012 2.03±0.002 Wavelength of detection, nm 290 290 Linearity range, µg/mL 0.5‐100 1‐50 Regression equation y = 7.0898 x ‐ 1.2818 y = 18.591 x ‐ 0.4337 Regression coefficient (r2) 1.000 0.9998 Sa, Standard deviation of intercept 1.0000 3.8277 Sb, Standard deviation of slope 0.0200 0.1264 Confidence limit for the slope 7.0898±0.0541 18.548±0.3514 Confidence limit for the intercept ‐1.2817±2.821 ‐0.4337±10.65 Limit of quantification, LOQ, μg/mL 0.2018 0.5980 Limit of detection, LOD, μg/mL 0.0665 0.1974 Drug in bulk 100.31±2.142 99.96±1.615 Recovery for Standard added 100.07±0.422 ‐ Recovery for drug in laboratory prepared mixtures 101.35±0.759 101.57±0.816 3. Results and discussion 3.1. Method development The core intention of this paper was to develop a stability indicating assay for OB with a short run time to allow for the high capacity of quality control laboratories. Thus, the conditions affecting the chromatographic performance were carefully studied in order to recognize the most suitable chromatographic system for determination of OB in presence of its degradation product, PABA. The choice was based on the highest number of theoretical plates and the best resolution between the two peaks. The selection of the mobile phase is of prime importance in the development of a chromatographic technique for proper elution, resolution, symmetrical peak shapes and reproducibility of the analytes [20]. Several mobile phases were evaluated using various proportions of different aqueous phases and organic modifiers. Methanol was tried as organic modifier; broad peaks with insufficient separation were obtained. Triethylamine and 1‐octane sulfonic acid sodium salt monohydrate as ion pairing reagents in the aqueous phase were tried to control the tailing but this did not give a satisfactory peaks tailing factor. The separation was investigated using mobile phases containing (30 and 40%) of acetonitrile with phosphate buffer with different pH’s (3.0, 4.5, and 6.5). It was found that peak skewing occurred with increasing the ratio of acetonitrile in the mobile phase while it had slight decreasing effect on the retention time. A mobile phase containing 70% acetonitrile was enough to give sharp symmetric peaks within a short runtime with reasonable resolution and sensitivity. The separation was investigated using mobile phases containing (50, 60, and 70 %) of phosphate buffer (pH = 3.0). It was found increasing the ratio of the buffer lead to longer run times with broad peaks, as phosphate buffer lead to inadequate resolution between OB and its degradation product, finally when acetate buffer was tried (30 %) pH = 2.35 with acetonitrile (70%), it gave sharp peak with shorter run time. The mobile phase was pumped at a flow rate of 0.8 mL/min which was suitable for good separation within a reasonable time. The chromatographic separation was tried using Discovery cyano column (Supelco) with dimension (15 cm × 4.6 mm, 5 µm) and Hypersil BDS C18 (15 cm × 4.6 mm, 5 µm). However, serious problems occurred in the determination of OB, in particular excessively tailed peaks, due to its quaternary ammonium nature. Inadequate resolution, broad peak and long retention time were obtained with the previous columns. But using Waters Atlantis C18 column (4.6 × 150 mm, 5 µm); less hydrophobic column showed better peak shapes within reasonable run time and satisfactory separation hence it became the column of choice for this study. Detection was tried at 224, 254 and 290 nm but finally 290 nm was chosen as it was found optimum for measurements. 3.2. System suitability tests The system suitability tests were performed to ensure that the proposed LC method was suitable to the analysis intended. The parameters of these tests are column efficiency (number of theoretical plates), tailing of chromatographic peak, repeatability as %R.S.D of peak area for six injections and reproducibility of retention as %R.S.D of retention time of a solution of a 50 µg/mL of OB (100% concentration) and 25 µg/mL of its degradant. The results of these tests for the proposed method are listed in Table 1. 3.3. Method validation 3.3.1. Linearity In this study, eight concentrations were chosen for OB and six concentrations were chosen for PABA. Each concentration was analyzed three times and linearity was studied for both drugs. A linear relationship between area under the peak (AUP) and concentrations (C) was obtained and the regression equation for OB and PABA was computed, Table 2. The linearity of the calibration curve was validated by the high value of correlation coefficient. 3.3.2. Accuracy Accuracy of the results was calculated by % recovery of 6 concentrations (injected in triplicates) of OB and PABA in bulk powder and in mixtures, and also by standard addition technique applied to Spasmomen® 40 mg. The results obtained are displayed in Table 2. 3.3.3. Precision The repeatability (intra‐day) was assessed by three determinations for each concentration (40, 50 and 60 µg/mL) for OB and (20, 25 and 30 µg/mL) for PABA representing 80, 100 and 120%, respectively. 100 El‐Kerdawy et al. / European Journal of Chemistry 7 (1) (2016) 97‐101 Table 3. Results for determination of repeatability of OB in powder and in presence of PABA by proposed LC method. Sample OB in powder OB in presence of PABA Concentration 40 μg/mL 80% 50 μg/mL 100% 60 μg/mL 120% 40 μg/mL 80% 50 μg/mL 100% 60 μg/mL 120% Mean 40.8473 51.0867 61.0862 40.8230 50.7244 59.6314 %Recovery 102.12 102.17 101.81 102.06 101.45 99.39 S.D. 0.1294 0.2262 0.2895 0.1141 0.2547 0.6080 %R.S.D 0.3168 0.4428 0.4739 0.2795 0.5021 1.0196 Table 4. Results for determination of repeatability of PABA in bulk powder and in presence of OB by proposed LC method. Sample PABA in powder PABA in presence of OB Concentration 20 μg/mL 80% 25 μg/mL 100% 30 μg/mL 120% 20 μg/mL 80% 25 μg/mL 100% 30 μg/mL 120% Mean 19.7837 24.7078 29.6433 19.8655 24.7974 29.4563 %Recovery 98.92 98.83 98.81 99.32 99.19 98.19 S.D. 0.0876 0.2096 0.2019 0.0706 0.1302 0.2587 %R.S.D 0.4429 0.8481 0.6809 0.3556 0.5251 0.8782 Table 5. Results for determination of inter‐day assay for OB in bulk powder and in presence of PABA by proposed LC method. Sample Inter‐day in bulk powder Inter‐day in presence of PABA Concentration 40 μg/mL 80% 50 μg/mL 100% 60 μg/mL 120% 40 μg/mL 80% 50 μg/mL 100% 60 μg/mL 120% Mean 40.49 50.99 60.39 40.74 50.30 59.76 %Recovery 101.23 101.98 100.65 101.85 100.60 99.60 S.D. 0.37 0.34 0.73 0.24 0.43 0.53 %R.S.D. 0.91 0.67 1.21 0.58 0.85 0.89 Table 6. Results for determination of inter‐day assay for PABA in bulk powder and in presence of OB by proposed LC method. Sample Inter‐day assay for PABA in bulk powder Inter‐day assay for PABA in presence of OB Concentration 20 μg/mL 80% 25 μg/mL 100% 30 μg/mL 120% 20 μg/mL 80% 25 μg/mL 100% 30 μg/mL 120% Mean 19.89 24.96 29.92 20.04 24.99 29.86 %Recovery 99.45 99.84 99.73 100.2 99.96 99.53 S.D. 0.13 0.20 0.38 0.20 0.21 0.41 %R.S.D 0.65 0.82 1.26 0.99 0.83 1.38 Table 7. Influence of mobile phase flow rate on resolution of OB in presence of PABA. 0.9 mL/min 0.8 mL/min0.7 mL/minFlow rate 14.08 14.6314.81 R, resolution factor Table 8. Influence of acetonitrile strength of mobile phase on resolution of OB from PABA. % of acetonitrile 68% 70% 72% R, resolution factor 14.25 14.63 14.24 Table 9. Influence of change of pH of mobile phase on resolution of OB. pH 2.25 2.35 2.45 R, resolution factor 14.45 14.63 14.66 The values of precision (%R.S.D.) of repeatability for both OB and PABA were found to be less than 1% in the three concentrations, (Table 3 and 4). Inter‐day precision was evaluated through replicate analysis of 40, 50 and 60 µg/mL and 20, 25 and 30 µg/mL for OB and PABA for three successive days. The values of precision (%R.S.D.) for OB and PABA were found to be less than 2% (Table 5 and 6). 3.3.4. Selectivity Selectivity is the ability of the analytical method to measure the analytes in the presence of interferences including degradation products or impurities, related substance and matrix components. In the present work, OB was determined and no chromatographic interference from any degradation products or excipients was found at the retention time of examined drug (Figure 3). In addition, the chromatogram of the sample solution was found identical to the chromatogram received by OB standard solution. These results demonstrate the absence of interference from other materials in the pharmaceutical formulation and therefore confirm the selectivity of proposed method. 3.3.5. Limit of detection and limit of quantification Limit of detection (LOD) and limit of quantification (LOQ) were calculated using following equation as per ICH guidelines [21]. LOD = 3.3 ×σ/S (1) LOQ = 10 ×σ /S (2) where σ is the standard deviation of response and S is the slope of the calibration curve, the results are shown in Table 2. 3.3.6. Robustness Robustness was performed by deliberately changing the chromatographic conditions. Only one factor was changed at a time while the other kept constant. The factor measured after each change was the resolution factor between the two peaks. Changing the flow rate of mobile phase by ±0.1, acetonitrile ratio by ±2%, Varying the pH of the mobile phase by ±0.1 didn’t have significant effect on chromatographic resolution by the proposed LC method for OB and its degradation product, indicating good robustness of the proposed method as shown in Tables 7‐9. El‐Kerdawy et al. / European Journal of Chemistry 7 (1) (2016) 97‐101 101 4. Conclusion The proposed LC method has the advantage of simplicity, precision, accuracy and convenience of separation and quantification of OB alone or in the presence of its expected degradation product in a reasonable analysis time. Moreover the proposed method is capable of simultaneous deter‐ mination OB and it’s degradant in laboratory prepared mixture and pharmaceutical formulation. Hence, the proposed LC method can be used for quality control of cited drug. 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