untitled ISSN 2 Novel ap for simu hydroch with kin Ahmed Ab 1 Pharmaceutica 2 Analytical Che * Corresponding Tel.: +2.02.2554 ARTICLE IN DOI: 10.5155/e Received: 11 M Received in rev Accepted: 02 Ju Published onlin Printed: 30 Sep KEYWORDS UPLC Olmesartan Hydrochlorothi Kinetic degrada Pharmaceutical Tandem mass s 1. Introduct Hyperten factor. The c prevention o drug treatm combination One of the m olmesartan m combination than either d ding to mon drug that i during abso selective AT (ARA‐II). OL 2H‐1,3‐dioxo 1‐({4‐[2‐(2H imidazole‐5‐ 6‐chloro‐1,1 sulfonamide, 2153‐2249 (Prin pproach f ultaneous hlorothiaz netic stud bdalazim Mo al Chemistry Depar mistry Department g author at: Pharm 41601. Fax: +2.02.2 FORMATION eurjchem.7.3.309- ay 2016 vised form: 21 June uly 2016 ne: 30 September 2 ptember 2016   S iazide ation l dosage form spectrometric tech tion nsion is a hig control of blood of cardiovascul ent may be no ns have been ad most successful medoxomil and n (FDC) [1,2]. T drug alone, and notherapy with s hydrolysed rption from th T1 subtype an M is described ol‐4‐yl)methyl4 H‐1,2,3,4‐tetrazo ‐carboxylate (F ‐dioxo‐3,4‐dihy , one of the E nt) / ISSN 2153‐ h Euro for UPLC‐ s determin zide in th dies of for ostafa 1,* and rtment, Faculty of P t, Faculty of Pharm aceutical Chemistry 5541601. E‐mail ad -314.1450 e 2016 2016 nique ghly prevalent d pressure (BP) lar morbidity a ot overcome hy dded and appr combinations i d hydrochloroth This combinati d is effective in h either agent to active met he gastrointesti ngiotensin II chemically as t 4‐(2‐hydroxypro ol‐5‐yl)phenyl]p igure 1). On th ydro‐2H‐1,2,4‐b oldest and w uropean Journal Europ 2257 (Online)  http://dx.doi.org/1 pean Jo Journal we ‐ESI‐Tand nation of eir pharm ced degra d Maha Mah Pharmacy, Helwan macy, Helwan Unive ry Department, Fac ddress: ahmead34@ ABSTRACT A simple, rob spectrometric olmesartan m pharmaceutica 50×2.1 mm (1 and 0.1% form reaction moni electrospray io and 3.0‐50.0 n acceptable. Th limits of quan method was pharmaceutica degradation of ICH guidelines reported meth method in qua Cite this: Eur. cardiovascular ) is important f and mortality. ypertension. Se roved in the m is the combinat hiazide in fixed on is more eff patients not re [3,4]. OLM is a tabolite, olmes inal tract [5]. I receptor antag the (5‐methyl‐2 opan‐2‐yl)‐2‐pr phenyl}methyl) e other hand, H benzothiadiazin widely used th l of Chemistry 7 pean Journal of C 2016 Atlanta Pub 10.5155/eurjchem ournal ebpage: www dem mass olmesart maceutica adation hmoud Abou University, 11795, ersity, 11795, Cairo, culty of Pharmacy, H @yahoo.com (A.A. M bust ultra‐perfor (UPLC‐MS/MS) medoxomil (OLM al formulation. 1.9 μm) column, mic acid aqueous itoring (SRM) m onization (ESI). ng/mL for OLM a he lower limits o ntitation were 1 successfully ap al dosage form f both drugs und s and there is no hod. The simpli ality control of th J. Chem. 2016, 7 r risk for the Single everal market. tion of d dose fective espon‐ a pro‐ sartan It is a gonist 2‐oxo‐ ropyl‐ )‐1H‐ HCT is ne‐7‐ iazide diur form trea met min pin HPT dete form emu mas plas and con dev [10 mod mod pre (3) (2016) 309‐ Chemistry blishing House LL m.7.3.309-314.14 of Che w.eurjchem.co s spectrom tan medox al combina u‐El Alamin Cairo, Egypt , Egypt Helwan University, Mostafa). rmance liquid c technique was M) and hydrochlo Chromatograph with gradient e s solution. Detec mode on a triple Linearity was a and HCT, respec of detection were 1.900 and 1.767 pplied for deter m. The method der different con o significance dif icity and sensit he cited drugs. 7(3), 309‐314 retics (Figure 1 m of ARA‐II atment and man Our literature thods have be nation of combi e Besylate [6‐ TLC [9,10] an ermination of m [11‐13] and ulsion oil, solv ss have been u sma [15] and f d HCT [16]. The nditions is not voiced from ,12,17‐19]. We use select de to obtain h de, the intact d sence of impur ‐314 LC ‐ All rights rese 450 emistry om metric me xomil and ation 2 11795, Cairo, Egyp chromatography developed for th orothiazide (HC hic separation lution of mobile ction of analytes e quadrupole m chieved over co ctively. Intra and e found to be 0.6 7 ng/mL for OL rmination the c was applied t nditions. The me fference betwee tivity of this m 1). More recent and hydrochl nagement of hy survey reveale een done by s ination of OLM 8]. Several chr d HPLC‐UV or both drugs in for determinat vents and surfa used for determ for pharmacok e stability of OL fully studied. M the kinetic p tive reaction m high selectivity drug can be eas ities and/or de erved ‐ Printed in y ethod d pt. y coupled to ES he simultaneous CT) in bulk and was done on a e phase consiste s was carried ou ass spectromete ncentration ran d inter day repro 627 and 0.583 ng LM and HCT, re cited drugs in o study the ki thod was validat n the proposed ethod allows th tly, a new comb lorothiazide is ypertension. d that there are pectrophotome and HCT (FDC romatographic r HPLC‐PDA w their pharmac tion of OLM sol actant [14]. Als mination both d inetic studies o LM and/or HCT Most of the pu parameters o monitoring Tand y of separation ily detected an gradation prod n the USA SI‐Tandem mass s quantitation of their combined a Hypersil gold d of acetonitrile t using selective er coupled with ges of 2.0‐200.0 oducibility were g/mL and lower espectively. The their combined inetic of forced ted according to method and the he utilization of bination dosage s indicated in e few analytical etry for deter‐ C) with amlodi‐ techniques as were used for ceutical dosage lubility in nano so, LC‐Tandem drugs in human of FDC of OLM T under forced blished papers f degradation dem mass scan n. In this scan nd quantified in ducts [20,21]. s f d d e e h 0 e r e d d o e f e n l ‐ ‐ s r e o m n M d s n n n n 310 Mostafa and Alamin / European Journal of Chemistry 7 (3) (2016) 309‐314 (a) (b) (c) Figure 1. Chemical structures of olmesartanmedoxomil (OLM) (a), hydro chlorothiazide (HCT) (b), and diphenhydramine (IS) (c). These scan mode allow us to use our method as stability indicating method. To our knowledge, there are no UPLC‐ MS/MS reported methods for simultaneous determination of both drugs in pharmaceutical preparations and/or kinetic forced degradation studies. Because of these two points, we tried to develop this novel UPLC‐MS/MS method of analysis and validate the method according to ICH guidelines [22]. 2. Experimental 2.1. Materials OLM (99.58%) and HCT (100.03%) were kindly supplied by the National Organization for Drug Control and Research (NODCAR) (Cairo, Egypt). Pharmaceutical dosage forms; Erastapex plus tablets (Multi‐Apex Pharma, Egypt) containing 20 mg OLM in combination with 12.5 mg HCT per tablet were obtained from local market. 2.2. Chemicals and reagents All chemicals used were of analytical grade and solvents were of HPLC grade. Diphenhydramine (IS), methanol, aceto‐ nitrile and formic acid were purchased from Sigma‐Aldrich, Germany. Sodium hydroxide, hydrochloric acid (32%, v:v) were purchased from El‐Nasr Company, Egypt. Pure deionized water was obtained by ElgaLabwater, Prima 7 (UK). 2.3. Instrumentations The analysis was achieved using a TSQ Quantum Access MAX triple stage quadrupole mass spectrometer, Thermo Scientific, New York, USA, equipped with an electrospray ionization (ESI) source. The control of the LC‐MS/MS system, acquisition and analysis of the data were performed utilizing Xcalibur software version 2.2. Chromatography was carried on Accela U‐HPLC system which was composed of Accela 1250 quaternary pump and Accela open autosampler, New York, USA (operated at 25 °C). 2.4. Chromatographic and mass spectrometric conditions Chromatographic separation was accomplished on HypersilGold column (C18‐bonded ultrapure silica based column) 50×2.1 mm (1.9 µm). Gradient elution was achieved using the binary mobile phase consisting of 0.1% formic acid aqueous solution (A) and acetonitrile (B) using a flow rate of 250 µL/min, where elution was performed at room tempera‐ ture. A gradient program was conducted as follows: 20 % B at zero time then ramped to 90% B from 0.0‐1.5 min, hold at 90% B till 3 min, back to 20% B from 3.0‐5.0 min. The injection volume was 5 µL and the total run time for each sample was 5 min. The mass spectrometric detection method was carried out in the positive‐ion mode for OLM and IS but negative mode for HCT utilizing electrospray ionization (ESI) and selected reaction monitoring mode. The optimized parameters are: auxiliary gas of 5 psi, sheath gas of 25 psi, capillary tempera‐ ture of 270 °C, turbo ion spray temperature of 400 °C and ion spray voltage of 3600 V. The quadrupole mass spectrometer was operated at the SRM mode, monitoring the transition of molecular ions to the product ions for OLM (m/z) 559.06 → 206.08, HCT (m/z) 296.90 → 53.70 and IS (m/z) 256.20 → 167.16. The collision energies were 29, 14 and 14 eV for OLM, HCT and IS, respectively. 2.5. Standard solutions Stock standard solutions of 0.1 mg/mL for OLM, HCT and IS were prepared in methanol and stored at 4 °C. Further dilution of each stock standard solution was made using methanol to obtain the appropriate working standard solu‐ tions which were also stored at 4 °C. 2.6. Procedures 2.6.1. Construction of calibration curves Standard calibration solutions were prepared from the working standard solutions of each drug. These calibration solutions of each drug in the concentration ranges of 2.0‐200.0 ng/mL for OLM and 3.0‐50.0 ng/mL for HCT. Each of the calibration solutions had a concentration of 5 ng/mL of IS. A volume of 5 μL of each solution was injected into the LC‐ MS/MS system. For each drug, a calibration curve was constructed by plotting the ratios of its peak area to IS peak areas versus the corresponding concentrations. 2.6.2. Laboratory prepared mixtures The working standard solutions of each of the two drugs were mixed in different ratios to obtain binary solutions of OLM and HCT in the concentration range of 2.0‐200.0 and 3.0‐ 50.0 ng/mL, respectively, then 5 ng/mL IS was added in each solution. An aliquot of 5 μL of each solution was injected into the LC‐MS/MS system. The percentage recoveries were calcu‐ lated by means of the corresponding regression equations or from the calibration graphs. 2.6.3. Analysis of pharmaceutical dosage form Ten tablets contain both analysts were accurately weighed and finely powdered. An accurate amount claimed to one table was ultrasonicated with methanol for 20 min then filtration. Complete the volume to 100 mL with methanol. Solutions containing the nominated range concentration were prepared and the procedure was continued as described under the procedure in Section 2.6.1. The percentage recoveries were calculated b equations or 2.6.4. Sampl Forced d were carried separately to NaOH. The s 105 °C for d then neutral methanol. Th the procedur 3. Results an 3.1. Method The pres OLM and IS e in the form o in case of OL by adjusting through the sheath gas intensity du spray voltage cone of mass V for the pos 3000 V for n attract the io The opt product ion 296.90 → 53 these LC‐MS and IS were tographic co and separat columns we hypersilGold 0.1% formi acetonitrile a 0.1% formi pressure. It the precent o drug. Represen For obtainin characteristi selection of Chromatogra adequate re elution wit acetonitrile. Figure 2. Repr internal standa by means of r from the calibr le degradation degradation stu d out in which o 10 mL of 1.0 solution was he different time in lized [19]. The he procedure w re in Section 2.6 nd discussion development sence of acidic enhance of the of [M‐H]‐ and p LM and IS. The g sheath gas t orifice of mass more than th e to dispersion e was responsib s analyser. It wa sitive mode (in negative mode ons toward mas timized SRM t m/z) are OLM 3.70 and IS (m S/MS conditions e 2.28, 0.73 an nditions were o tion of each a ere test and d. Different rati c acid were and 0.1% form c acid. Also, was found tha of organic mob ntative chroma ng good chrom ics for the su f the mobile aphic separatio etention times th 0.1% form resentative SRM ch ard (IS). Mostafa and Ala f the corresp ration graphs. n dy under differ 10 mg of each N alcoholic HC eated in thermo ntervals 15, 30 e prepared sol was continued 6.1. moiety in HCT ionization of H positive mode in e intensity of pe that helps in i s analyser, how he required v n of the ions. ble for attractin as found that vo n case of OLM a (in case of HCT ss analyser. transitions (pre (m/z) 559.06 → m/z) 256.20 → s, the retention nd 2.11 min, re optimized to ob analyte and IS. the most ef o of methanol used. The se mic acid is more methanol inc at gradient elu ile phase was a atograms were matographic se ubsequent qua phase was an on of the analyte and peak sha mic acid aque hromatograms of O amin / European ponding regre rent stress cond h drug was subj Cl or 2.0 N alc ostatic water b 0, 45, 60 and 9 lutions diluted as described T and basic moi HCT in negative n the form of [M eaks were optim ntroducing the wever increasin value decrease On the other ng the ion towa oltage less than and IS) and less T) will not enou ecursor ion m → 206.08, HCT 167.16. By app n times of OLM espectively. Ch btain high reso . Different rev fficient one is and acetonitril eparation pow e than methano rease column tion with incre able to separate shown in Figu eparation and antitative work n important f es was achieved apes using gra eous solution OLM, HCT in pres n Journal of Chem ession ditions jected oholic ath at 0 min d with under iety in mode M+H]+ mized e ions ng the d the hand, rd the n 3600 s than ugh to m/z → (m/z) plying M, HCT roma‐ lution versed s the le and wer of ol and back easing e each ure 2. peak k, the factor. d with adient and ence of the ace befo chr for 3.2. test qua 3.2. sep rela bot line ng/ coe regr ana OLM HCT whe dru valu line Tab MS/ Para Line Slop Inte r2 LOD LOQ 3.2. (LO ana (LO qua don slop LOD LOQ whe resp blan regr regr pro inte 3.2. mad solu mistry 7 (3) (2016 The presence sharpness of tonitrile helped ore 4 min at omatographic quantitative wo . Method valida The validity o ted in terms of antification, acc .1. Linearity an By applying th aration and ationship betw h drugs was fo ear in the conc /mL for OLM fficients for b ression parame alysis of the data M: A = 0.005 T: A = 1.532 ere A is the rela ug in ng/mL an ues of the corr earity of the cali le 1. Regression /MS method. ameter earity range (ng/m pe (b) ercept (a) D (ng/mL) Q (ng/mL) .2. Limit of qua OD) The limit of de alyte that can e OQ) is the low antified by the ne base on stan pe of calibration D = 3.3 σ/s Q = 10 σ/s ere, s = Slope ponse. Residual (S.D.) nk response ression line (y ression line Sy oposed method ercept. The resu .3. Accuracy Evaluation of de by the anal ution of each dr 6) 309‐314 of formic acid i f peaks. Rapid d in early elut a flow rate conditions, the ork even at very ation of the propose linearity, range uracy and prec nd range he optimum co mass spectro ween concentrat ound. The calib centration rang and HCT, r both curves ar eters are listed a gave the follo 545 + 0.00498 205 + 2.84363 ative peak areas nd r2 is the reg relation coeffic ibration graphs parameters for O O mL) 2 0 0 0 0 1 antitation (LOQ etection (LOD) easily detect, w west concentra method. Calcu ndard deviatio n curve [23] (Ta of calibration ) of response co or residual (y‐residual) or /x, (Standard e calculation wa ults were listed the accuracy o lysis of five co rug each concen in the mobile p d rise in the tion of the two of 250 μL/mi e peak shape w y low concentra ed UPLC‐MS/M es, limits of dete cision. nditions for ch ometric detect tion and peak ration curve w ges of 2.0‐200. respectively. T re greater tha in Table 1. Lin wing equations × C (r2 = 0.99 × C (r2 = 0.99 s and C is the co gression coeffic cients (>0.999) s. OLM and HCT by OLM .0‐200.0 .005 .005 .9997 .627 .90 Q) and limit of is the lowest co while the limit o tion of analyt ulations of LOD on (S.D.) of the able 1). n curve, σ = r ould be calculat standard dev r S.D. of y‐int error of estima as done based in Table 1. of the propose oncentrations o ntration repeate 311 phase improves proportion of o drugs and IS in. With these was satisfactory ations. S method was ection, limits of hromatographic tion, a linear area ratio for was found to be 0 and 3.0‐50.0 The regression an 0.999. The near regression s: 997) (1) 992) (2) oncentration of cient. The high indicate good the proposed LC‐ HCT 3.0‐50.0 2.844 1.532 0.9992 0.583 1.767 f detection oncentration of of quantitation te that can be D or LOQ were e response and (3) (4) esidual S.D. of ted from S.D. of viation of the tercept of the te) [23]. In the on S.D. of the ed method was of the standard ed three times. s f S e y s f c r r e 0 n e n f h d ‐ f n e e d f f e e e e s d 312 Mostafa and Alamin / European Journal of Chemistry 7 (3) (2016) 309‐314 Table 2. Data of accuracy and precision obtained by the proposed method and the reported ones [3] for the analysis of OLM and HCT in pure form. Item OLM HCT Proposed Reported Proposed Reported Mean±S.D. 100.75±0.78 99.84±0.85 99.87±0.93 100.53±0.7 % R.S.D. 0.77 0.85 0.93 0.70 n 5 5 5 5 % Error (% R.S.D./√n) 0.344 0.379 0.415 0.313 Variance 0.61 0.72 0.86 0.49 t‐test (2.31) * 1.25 0.27 F‐test (5.409) * 1.18 1.76 Intra‐day precision 99.93±0.85 100.15±0.92 Inter‐day precision 99.80±0.83 100.51±1.21 S.D.: Standard deviation; %R.S.D.: Percent relative standard deviation; Values in parenthesis tabulated values at p = 0.05. Table 3. Results of system suitability of the proposed method. Compound RT (min) Capacity factor (k) Selectivity (α) Resolution (Rs) Tailing factor Theoretical plates HETP OLM 2.28 4.56 ‐ ‐ 1.15 3556 0.005 HCT 0.73 5.2 1.22 1.32 1.23 2250 0.025 Table 4. Determination of OLM and HCT in laboratory prepared mixtures by the proposed method. Concentration (ng/mL) % Recovery OLM HCT OLM HCT 5 5 100.52 98.56 20 10 99.52 100.77 25 15 100.42 98.72 30 20 100.18 100.03 40 25 99.15 99.12 80 50 99.24 99.29 Mean±S.D. 99.84±0.61 99.42±0.84 % R.S.D. 0.61 0.845 Variance 0.31 0.59 Table 5. Assay of OLM and HCT in their combined tablets using proposed LC‐MS/MS and reported methods [11]. Item % Recovery Proposed Reported [11] OLM HCT OLM HCT Mean±S.D. 100.24±0.93 98.84±0.85 99.60±0.83 99.52±0.59 % R.S.D. 0.93 0.85 0.83 0.59 Variance 0.86 0.72 0.69 0.35 t‐test (2.12) * 0.56 1.1 F test (3.787) * 1.25 2.06 * Values in parentheses are the tabulated values at p = 0.05 (n = 5). The results of the proposed method were compared with those obtained from reference methods [11]. Statistical comparison between the proposed method and reported method of both drugs was showed that there was no significant difference in their accuracy and precision as shown by the results of student´s t‐test and variance ratio F‐test, respectively (Table 2). 3.2.4. Precision Evaluation of the intra‐day precision was made by replicate assay of the standard solutions of the studied drugs on the same day, while the inter‐day precision was evaluated through replicate the assay of standard solutions of the studied drugs on three successive days (Table 2). The value of standard deviation (S.D.) was small what indicates that the repeatability of the proposed method is good. 3.2.5. System suitability System suitability applied to confirm the suitability of chromatographic system for analysis with high agrees of accuracy and precision. Following the USP guidelines [24] and with concordance with the parameters value [25]. The suitability of method was done by determination of analytes concentration using external method (Table 3). 3.2.6. Robustness of the method The robustness of an analytical method measures the capacity of the method to restrain minute but deliberate changes in method parameters [26]. Evaluation of the robustness of the proposed method was done for the chromatographic parameters as well as, the mass parameters, e.g. flow rate of mobile phase (±10 μL/min), vaporizer tempe‐ rature or transfer capillary temperature (±5 °C), collision energy (±2 V) and sheath gas pressure (±5 psi).The changes in theses parameters did not show significant changes in the values of peak areas. 3.3. Application of the proposed method The proposed method was applied for analysis laboratory mixture of OLM and HCT in different proportions. Satisfactory results were obtained and listed in Table 4. Erastapex plus tablets were analysed using our proposed LC‐MS/MS method to demonstrate its suitability to analysis both drugs in their pharmaceutical formulation without interference from the tablet additives and for quality control purpose. The concentration of each drug was calculated from its regression equation (Table 5). 3.4. Kinetic forced degradation During storage of drugs, it affect by different conditions such as temperature, pH of solution, light, oxidation. These conditions will enhance the degradation of pharmaceutical products during storage. The cited drugs were subjected to acidic (1.0 N alcoholic HCl) and alkaline (2.0 N alcoholic NaOH) degradation. The degradation products of OLM and HCT in acidic and basic conditions were identified by LC‐MS/MS (Figure 3 and 4). OLM und olmesartan 447.35 (Figu with m/z = product wer 205.92 due t suggestion f and HCT wer kinetics of a were investi different tim limiting facto between cha degradations concentratio follows zero Because reagents, the dergoes hydroly free acid whic ure 5). Anothe 402.61 (Schem re identified b to loss of HCN a for identificatio re confirmed b acidic and alkal igated by draw me intervals (1 or in determina anges in concen s depend on on by time. Mo order, first ord OLM and HCT e degradation o Mostafa and Ala Figure 3 Figure 4 ysis under forc ch identified i er major produ me 1). In case by Q1 scan wi and SO2, respect on of degradati y the reported line degradatio wing the conce 15, 30, 45, 60 ation of reactio ntration by time relations be ost of pharmac der or pseudo‐o were place wi of both drugs w amin / European 3. Full scan spectra 4. Full scan spectra ed condition to n the Q1 with ucts were iden of HCT, two m ith m/z 269.21 tively (Figure 6 on products of articles [27,28 ons of OLM and entration of dr 0 and 90 min) n rate is the re e. The orders of etween change euticals degrad rder [29]. ith a large volu was showed ps n Journal of Chem a of [M+H]+ of the d a of [M‐H]+ of the d o form h m/z ntified majors 1 and 6). Our f OLM 8]. The d HCT rug at ). The elation f these es in dation ume of seudo‐ firs occ Any will min deg rem left con from t in T mistry 7 (3) (2016 degradation produ degradation produ t order kinet urred due to p y change in con l be negligible i nor reactants ( gradation param maining concent for 50% con ncentration (t90 m the following = . t = . The results of Table 6. 6) 309‐314 ucts of OLM. ucts of HCT. tics [30]. Pse presence of two ncentration of m in comparison drugs). The rat meters were o tration against ncentration (t1 0) for each forc g equation 5 [30 degradation ki eudo‐first‐orde o reactants in major reactants to change in co te of degradati btained by plo time. Rate con 1/2) and time ced condition 0]. netic paramete 313 r degradation different ratio. s (acid or base) oncentration of ion and kinetic otting of log % nstant (K), time left for 90% were obtained (5) ers were shown n . ) f c % e % d n 314 Mostafa and Alamin / European Journal of Chemistry 7 (3) (2016) 309‐314 Table 6. Summary of degradation kinetic parameters for pseudo‐first order reaction. Items AMO ASN Acid Alkaline Peroxide Acid Alkaline Peroxide r2 0.9303 0.9225 0.9730 0.8930 0.9254 0.9355 K ((ng/mL).min‐1) ‐0.0180 ‐0.0095 ‐0.0240 ‐0.0070 ‐0.0250 ‐0.0050 t1/2 (min) 38.33 72.63 28.75 98.57 27.60 138.00 t90 (min) 15 4.2 21 5.8 11.05 4.38 Figure 5. The proposed structures of the main degradation products of OLM. Figure 6. The proposed structures of the main degradation products of HCT. 4. Conclusion As conclusion, we developed and validated a new ULPC‐ MS/MS method for simultaneous determination of OLM and HCT in pharmaceutical dosage form. The utilization of UPLC improves peak resolutions and separation in short time to save time and solvents. The method is simple, rapid, selective and sensitive. The proposed method was suitable for routine analysis and quality control testing of combined mixtures of both drugs in pharmaceutical dosage forms. The stability of both drugs in acidic and alkaline medium were studied revealed that both OLM and HCT are easily degraded in the tested mediums. Some of the degradation products of OLM and HCT were identified by LC‐MS. Acknowledgement Authors acknowledge The Pharmaceutical Services Centre and LC‐MS Unit, Faculty of Pharmacy, Helwan University, Cairo, Egypt for affording the facilities during method development. References [1]. Bramlage, P.; Zemmrich, C.; Ketelhut, R.; Wolf, W. P.; Fronk, E. M.; Schmieder, R. E. Vasc. Health Risk Manag. 2013, 9, 475‐483. [2]. Mohan, J. C.; Jain, R.; Chamle, V.; Bhargava, A. J. Clin. Diagn. Res. 2015, 9(8), OC10‐3. [3]. Chrysant, S. G.; Weber, M. A.; Wang, A. C.; Hinman, D. J. Am. Soc. Hypertens 2004, 17, 252‐259. [4]. Sellin, L.; Stegbauer, J.; Laeis, P.; Rump, L. C. J. Hyperten. 2005, 23, 2083‐2092. [5]. Laeis, P.; Püchler, K.; Kirch, W. J. Hypertens Suppl. 2001, 19(1), S21‐ 32. [6]. Rote, A. R.; Bari, P. D. Indian J. Pharm. Sci. 2010, 72, 111‐113. [7]. Merey, H. A.; Ramadan, N. K.; Diab, S. S.; Moustafa, A. A. Spectrochim. Acta A 2014, 125, 138‐146. [8]. Darwish, H. W. Chem. Cent. J. 2013, 7, 22‐22. [9]. Solanki, T. B.; Shah, P. A.; Patel, K. G. Indian J. Pharm. Sci. 2014, 76, 179‐187. [10]. Verma, P. K.; Kamboj, V. K. Pak. J. Pharm. Sci. 2013, 26(1), 209‐215. [11]. Vidyadhara, S.; Reddyvalam, L. C. S.; Rao, B. V.; Tejaswi, K.; Reshma, M. Oriental J. Chem. 2014, 30(1), 195‐201. [12]. Yunoos, M.; Sankar, D. G. Int. J. Pharm. Sci. Drug. Res. 2015, 7(3), 290‐ 297. [13]. Srikanth, R. R.; Murali, K. R.; Vekaria, N. A.; Rao, S. V.; Mantena, B. P. V. J. Chromatogr. B. 2015, 38, 1343‐1354. [14]. Gorain, B.; Choudhury, H.; Biswas, E.; Barik, A.; Jaisankar, P.; Pal, T. K. RSC Advances 2013, 3, 10887‐10893. [15]. Kumar, A.; Verma, P. R. P.; Monif, T.; Khuroo, A. H.; Iyer, S. S.; Singh, A. K. J. Chromatogr. B. 2012, 35, 59‐78. [16]. Chae, J. W.; Baek, I. H.; Seo, J. W.; Jung, S. H.; Back, H. M.; Song, B. J.; Lee, B. Y.; Yun, H. Y.; Kang, W.; Kwon, K. I. Int. J. Clin. Pharmacol. Ther. 2014, 52(8), 676‐683. [17]. Ebeid, W. M.; Elkady, E. F.; El‐Zaher, A. A.; El‐Bagary, R. I.; Patonay, G. Anal. Bioanal. Chem. 2014, 406, 6701‐6712. [18]. Kumar, K. K.; Rao, C. K.; Madhusudan, G.; Mukkanti, K. Am. J. Analyt. Chem. 2012, 3, 50‐58. [19]. Jain, P. S.; Patel, M. K.; Gorle, A. P.; Chaudhari, A. J.; Surana, S. J. J. Chromatogr. Sci. 2012, 50, 680‐687. [20]. Mowaka, S.; Mohamed, D. RSC Advances 2015, 5, 60467‐60481. [21]. Mohamed, D; Mowaka, S.; Mostafa, A. Eur. J. Chem. 2014, 5, 181‐85. [22]. ICH Harmonized Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology, Q2(R1), Current Step 4 Version, Parent Guidelines on Methodology, 2005. [23]. Shrivastava, A.; Gupta, V. Methods for the determination of limit of detection and limit of quantitation of the analytical methods, 2011; Vol. 2. [24]. El‐Gizawy, S. M.; Abdelmageed, O. H.; Omar, M. A.; Deryea, S. M.; Abdel‐Megied, A. M. Am. J. Analyt. Chem. 2012, 3, 422‐430. [25]. Adamovics, J. A. Chromatographic analysis of pharmaceuticals; Marcel Dekker Inc. , New York, 1997. [26]. ICH Harmonized Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology, Q2 (R1), Current Step 4 Version, Parent Guidelines on Methodology Dated November 6; 1996, incorporated in November 2005. [27]. Murakami, T.; Konno, H.; Fukutsu, N.; Onodera, M.; Kawasaki, T.; Kusu, F. J. Pharm. Biomed. Anal. 2008, 47, 553‐559. [28]. Mahajan, A. A.; Thaker, A. K.; Mohanraj, K. J. Brazilian Chem. Soc. 2012, 23, 445‐452. [29]. Gorog, S. J. Pharm. Biomed. Anal. 2008, 48, 247‐253. [30]. Florence, D. A. A. T. Physicochemical Principles of Pharmacy, Macmillan Press, London 1998.