Amlodipine, Olmesartan and Hydrochlorothiazide..

Transcription

1 PART - A SECTION-II HPLC METHOD DEVELOPMENT AND VALIDATION OF COMBINED DOSAGE FORM OF AMLODIPINE BESYLATE, OLMESARTAN MEDOXOMIL AND HYDROCHLOROTHIAZIDE IN TABLETS

2 SECTION II: HPLC METHOD DEVELOPMENT AND VALIDATION FORSIMULTANEOUS DETERMINATION OF AMLODIPINE BESYLATE, OLMESARTAN MEDOXOMIL AND HYDROCHLOROTHIAZIDE IN TABLETS 1. INTRODUCTION OF DRUGS 1.1 Introduction of Amlodipine Besylate Amlodipine is a long-acting 1,4-dihydropyridine calcium channel blocker. It acts primarily on vascular smooth muscle cells by stabilizing voltage-gated L-type calcium channels in their inactive conformation. By inhibiting the influx of calcium in smooth muscle cells, Amlodipine prevents calcium-dependent myocyte contraction and vasoconstriction. A second proposed mechanism for the drug s vasodilatory effects involves ph-dependent inhibition of calcium influx via inhibition of smooth muscle carbonic anhydrase. Some studies have shown that Amlodipine also exerts inhibitory effects on voltage-gated N-type calcium channels. N-type calcium channels located in the central nervous system may be involved in nociceptive signaling and pain sensation. Amlodipine is used to treat hypertension and chronic stable angina [1] Description Amlodipine besylate is chemically described as 3-Ethyl-5-methyl (±)-2-[(2- aminoethoxy) methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridine dicarboxylate, monobenzenesulphonate. Its empirical formula is C 20 H 25 CIN 2 O 5 C 6 H 6 O 3 S, and its structural formula is as in figure 1. O O Cl H N O O O NH 2 OH O S O Figure 1: Structure of Amlodipine besylate Amlodipine besylate is a white crystalline powder with a molecular weight of 567.1gm/mole. 74

3 1.1.2 Pharmacodynamics Amlodipine belongs to the dihydropyridine (DHP) class of calcium channel blockers (CCBs), the most widely used class of CCBs. There are at least five different types of calcium channels in Homo sapiens: L-, N-, P/Q-, R- and T-type. It was widely accepted that DHP CCBs target L-type calcium channels, the major channel in muscle cells that mediate contraction; however, some studies have indicated that Amlodipine also binds to and inhibits N-type calcium channels (see references in Targets section). Similar to other DHP CCBs, Amlodipine binds directly to inactive L-type calcium channels stabilizing their inactive conformation. Since arterial smooth muscle depolarizations are longer in duration than cardiac muscle depolarizations, inactive channels are more prevalent in smooth muscle cells. Alternative splicing of the alpha-1 subunit of the channel gives Amlodipine additional arterial selectivity. At therapeutic sub-toxic concentrations, Amlodipine has little effect on cardiac myocytes and conduction cells [2] Mechanism of action Amlodipine decreases arterial smooth muscle contractility and subsequent vasoconstriction by inhibiting the influx of calcium ions through L-type calcium channels. Calcium ions entering the cell through these channels bind to calmodulin. Calcium-bound calmodulin then binds to and activates myosin light chain kinase (MLCK). Activated MLCK catalyzes the phosphorylation of the regulatory light chain subunit of myosin, a key step in muscle contraction. Signal amplification is achieved by calcium-induced calcium release from the sarcoplasmic reticulum through ryanodine receptors. Inhibition of the initial influx of calcium decreases the contractile activity of arterial smooth muscle cells and results in vasodilation. The vasodilatory effects of Amlodipine result in an overall decrease in blood pressure. Amlodipine is a long-acting CCB that may be used to treat mild to moderate essential hypertension and exertionrelated angina (chronic stable angina). Another possible mechanism is that Amlodipine inhibits vascular smooth muscle carbonic anhydrase I activity causing cellular ph increases which may be involved in regulating intracelluar calcium influx through calcium channels. 75

4 1.1.4 Pharmacokinetics The metabolism and excretion of Amlodipine have been studied in healthy volunteers following oral administration of 14 C-labelled drug. Amlodipine is well absorbed by the oral route with a mean oral bioavailability of approximately 60%. Renal elimination is the major route of excretion with about 60% of an administered dose recovered in urine, largely as inactive pyridine metabolites. The major metabolite identified was 2-([4-(2-chlorophenyl)-3-ethoxycarbonyl-5-methoxycarbonyl-6-methyl-2- pyridyl]methoxy) acetic acid, and this represented 33% of urinary radioactivity. Amlodipine concentrations in plasma declined with a mean half-life of 33 h, while elimination of total drug-related material from plasma was slower. Amlodipine, a dihydropyridine calcium antagonist, was synthesized in an attempt to develop a compound with a pharmacokinetic profile characteristic of this class, which would also have an increased oral bioavailability and extended clearance time. A single intravenous dose of 10 mg resulted in an absolute bioavailability of 64% and a calculated elimination half-life of 34. The pharmacokinetic profile of oral doses showed similar changes. These results were significantly different from those seen with most other dihydropyridines (elimination half-life of 3 to 10 and absolute bioavailability of 10% to 30%) and nondihydropyridine calcium antagonists (elimination half-life 3 to 6 and low absolute bioavailability). With chronic oral dosing of Amlodipine once daily for 14 days, support was provided for the linearity of Amlodipine's pharmacokinetics and absence of such with chronic oral dosing with verapamil, diltiazem, and nifedipine. In the elderly population, elimination half-life of 5 mg oral doses is significantly prolonged suggesting decreased oral clearance or increased bioavailability. Comparison of the pharmacokinetics of Amlodipine in patients with chronic stable angina pectoris with the profile in healthy volunteers suggested that clearance is not altered in patients with chronic stable angina, steady state being reached 6 to 12 after administration of the drug. In patients with cirrhosis, elimination half-life is significantly prolonged suggesting that there is a greater accumulation of Amlodipine in patients with severe liver disease than in individuals with normal hepatic function [3]. 76

5 1.2 Introduction of Olmesartan Medoxomil Olmesartan is an antihypertensive agent which belongs to the class of medicines called angiotensin II receptor antagonists [4]. It acts rapidly to lower high blood pressure. It is marketed worldwide by Daiichi Sankyo, Ltd. and in the United States by Daiichi Sankyo, Inc. and Forest Laboratories Description Olmesartan medoxomil chemically (5-methyl-2-oxo-2H-1,3-dioxol-4-yl)methyl 4- (2-hydroxypropan-2-yl)-2-propyl-1-({4-[2-(2H-1,2,3,4-tetrazol-5-yl)phenyl]phenyl} methyl)-1h-imidazole-5-carboxylate (figure 2). Its molecular formula is C 29 H 30 N 6 O 6 having molecular mass gm/mole. N N N NH O CH 3 N O O O CH 3 N HO CH 3 O Figure 2: Structure of Olmesartan medoxomil Pharmacodynamics Olmesartan, a specific angiotensin II type 1 antagonist, is used alone or with other antihypertensive agents to treat hypertension. Unlike the angiotensin receptor antagonist losartan, Olmesartan does not have an active metabolite or possess uricosuric effects. Blockade of the angiotensin II receptor inhibits the negative regulatory feedback of angiotensin II on renin secretion, but the resulting increased plasma renin activity and circulating angiotensin II levels do not overcome the effect of Olmesartan on blood pressure [5] Mechanism of action Angiotensin II is formed from angiotensin I in a reaction catalyzed by angiotensin converting enzyme (ACE, kininase II). Angiotensin II is the principal pressor agent of the 77

6 renin-angiotensin system, with effects that include vasoconstriction, stimulation of synthesis and release of aldosterone, cardiac stimulation and renal reabsorption of sodium. Olmesartan blocks the vasoconstrictor effects of angiotensin II by selectively blocking the binding of angiotensin II to the AT 1 receptor in vascular smooth muscle. Its action is, therefore, independent of the pathways for angiotensin II synthesis. Olmesartan has more than a 12,500-fold greater affinity for the AT 1 receptor than for the AT 2 receptor[6] Pharmacokinetics Orally administered Olmesartan medoxomil was rapidly absorbed from the gastrointestinal tract and converted during absorption to Olmesartan, the pharmacologically active metabolite that was subsequently excreted without further metabolism. The medoxomil moiety was released as diacetyl that was rapidly cleared by further metabolism and excretion. Peak plasma concentrations of Olmesartan occurred 1-3 h after administration, after which concentrations decreased quickly. The elimination half-life was h. Olmesartan medoxomil was not measurable in plasma and excreta. The volume of distribution was low, consistent with limited extravascular tissue distribution. Bioavailability (C max and area under the curve) increased approximately in proportion with dose, after single and multiple daily oral doses, over the therapeutic dose range (up to mg daily), above which systemic availability of Olmesartan increased less than proportionally with increase in dose. Steady-state plasma concentrations of Olmesartan were reached within the first few daily oral doses. On average, approximately 40% of systemically available Olmesartan was excreted by the kidneys, the remainder being excreted in faeces, following secretion in bile. Renal clearance ( l/h) was independent of dose, accounting for approximately 9-12% of an oral dose. The absolute bioavailability of Olmesartan from Olmesartan medoxomil tablets was 28.6%. Olmesartan exhibited little or no binding to blood cells. No clinically significant steadystate pharmacokinetic interactions were observed following co-administration of Olmesartan medoxomil with digoxin, warfarin and aluminium magnesium hydroxide (antacid), supporting the low potential for clinically significant pharmacokinetic interactions to occur between Olmesartan medoxomil and co-administered drugs. 78

7 1.3 Introduction of Hydrochlorothiazide Hydrochlorothiazide, abbreviated HCTZ, HCT or HZT is a diuretic drug of the thiazide class that acts by inhibiting the kidneys' ability to retain water. This reduces the volume of the blood, decreasing blood return to the heart and thus cardiac output and, by other mechanisms, is believed to lower peripheral vascular resistance. Hydrochlorothiazide is a calcium-sparing diuretic, meaning it can help the body get rid of excess water while still keeping calcium [7] Description Hydrochlorothiazideischemically6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4,benzo thiadiazine-7-sulfonamide (Figure 3). Its molecular formula is C 7 H 8 N 3 O 4 S 2 Cl having molecular mass gm/mole. Cl H N H 2 N S S NH O O O O Figure 3: Structure of Hydrochlorothiazide Mechanism of action Hydrochlorothiazide belongs to the thiazide class of diuretics. It reduces blood volume by acting on the kidneys to reduce sodium (Na) reabsorption in the distal convoluted tubule. The major site of action in the nephron appears on an electroneutral Na+-Cl- co-transporter by competing for the chloride site on the transporter. By impairing Na transport in the distal convoluted tubule, Hydrochlorothiazide induces a natriuresis and concomitant water loss. Thiazides increase the reabsorption of calcium in this segment in a manner unrelated to sodium transport. Additionally, by other mechanisms, HCTZ is believed to lower peripheral vascular resistance [8] Pharmacokinetics Hydrochlorothiazide is not metabolized but is eliminated rapidly by the kidney. When plasma levels have been followed for at least 24, the plasma half-life has 79

8 been observed to vary between 5.6 and At least 61% of the oral dose is eliminated unchanged within 24. Hydrochlorothiazide crosses the placental but not the blood-brain barrier and is excreted in breast milk. Hydrochlorothiazide (hct) was administered orally in four different doses (12.5, 25, 50 and 75 mg), to eight healthy volunteers. Plasma and urine concentrations of hct were determined by GLC. Maximal plasma levels were found at h, and averaged 70, 142, 260 and 376 ng/ml respectively. The peak plasma levels and AUC 0 9h of hct were highly correlated (p<0.001) with the dose. The decline in the plasma curve was biphasic in those experiments in which the plasma levels of hct could be determined for at least 24 h. The half life of the slower phase lay between 5.6 and 14.8 h. The urinary recovery of hct, which represented the gastrointestinal absorption, averaged 65 to 72 per cent of the dose. The mean renal plasma clearance did not vary with the dose and averaged 319 to 345 ml min 1. The diuresis during the 10 h after hct 12.5 mg exceeded that after placebo by a mean of 800 ml. The diureses was not increased further after higher doses of hct. The maximal natriuretic effect (+ 100 mmol), too, was found after the 12.5 mg dose. The excretion of potassium, however, rose with increasing doses; the maximal increment, after 75 mg hct, averaged 25 mmol. The excretion of calcium was significantly increased after 50 mg hct (+ 0.6 mmol). The maximal effect on magnesium excretion occurred after 25 mg hct (+ 0.5 mmol). In healthy volunteers there was no correlation between peak plasma level of hct or AUC 0 9h and the renal excretion of water and electrolytes [9]. 80

9 2. LITERATURE REVIEW The literature reviews suggest that various analytical methods were reported for determination of Amlodipine, Olmesartan and Hydrochlorothiazide as drug, in pharmaceutical formulation and in various biological fluids. The literature reviews for analysis of all three drugs are as under: 1. Yu, Yong-Jie; Wu, Hai-Long; Niu, Jing-Fang; Zhao, Juan; Li, Yuan-Na; Kang, Chao; Yu, Ru-Q highlighted a strategy that employs a new time shift alignment method derived from the known Rank Minimization method for aligning chromatographic peak shifts among samples and then uses trilinear decomposition methodology to interpret the overlapped chromatographic peaks to quantify analytes of interest. The performance of this novel strategy for chromatographic data analysis was evaluated using simulated chromatographic data as well as real chromatographic data. The new time shift alignment method can accurately correct time shifts in test samples even in the presence of unexpected interferences, and thus the low-rank trilinearity of the same analyte can be obtained, which will be helpful for trilinear decomposition to achieve the 2nd-order advantage [10]. 2. DarwishHany W. have reported a new, simple and specific spectrophotometric methods and artificial neural network (ANN) in accordance with ICH guidelines for the simultaneous estimation of Olmesartan (OLM), Amlodipine (AML), and Hydrochlorothiazide (HCT) in commercial tablets. For spectrophotometric methods, Amlodipine (AML) was determined by direct spectrophotometry at 359 nm and by application of the ratio subtraction, the AML spectrum was removed from the mixture spectra. Then Hydrochlorothiazide (HCT) was determined directly at 315 nm without interference from Olmesartan medoxamil (OLM) which could be determined using the isoabsorptive method. The calibration curve is linear over the concentration range of , and mug ml-1 for AML, OLM and HCT, respectively. ANN (as a multivariate calibration method) was also applied for the simultaneous determination of the three analytes in their combined pharmaceutical dosage form using spectral region from nm. The proposed methods were successfully applied for the assay of the three analytes in laboratory prepared mixtures and combined pharmaceutical tablets with excellent recoveries. No interference was observed from common pharmaceutical additives. The results were favorably compared with those obtained by a reference 81

10 spectrophotometric method. The methods are validated according to the ICH guidelines and accuracy, precision and repeatability are found to be within the acceptable limit [11]. 3. Shentu, Jianzhong: Fu, Lizhi; Zhou, Huili; Hu, Xing Jiang; Liu, Jian; Chen, Junchun; Wu, Guolan have reported an automated method (XLC-MS/MS) that uses online solid-phase extraction-coupled with HPLC-tandem mass spectrometry to quantify Amlodipine in human plasma. Automated pre-purification of plasma was performed using 10 mm 2 mm HySphere C8 EC-SE online solid-phase extraction cartridges. After being eluted from the cartridge, the analyte and the internal standard were separated by HPLC and detected by tandem mass spectrometry. Mass spectrometric detection was achieved in the multiple reaction monitoring mode using a quadrupole tandem mass spectrometer in the positive electrospray ionization mode. The XLC-MS/MS method was validated and yielded excellent specificity [12]. 4. Patel, Samixa R.; Patel, Chhaganbhai N. have developed a simple, accurate, precise, economical, and reproducible spectrophotometric method for simultaneous determination of Olmesartan, Amlodipine and Hydrochlorothiazide in combined pharmaceutical dosage forms. The excipients in the com. tablet preparation did not interfere with the assay. The λmax for Olmesartan, Amlodipine and Hydrochlorothiazide were 252 nm, 360 nm and 271 nm respectively. At 360 nm, Amlodipine showed some absorbance while Olmesartan and Hydrochlorothiazide showed zero absorbance so that Amlodipine was estimated at 360 nm. While at 252 nm and 271 nm Olmesartan and Hydrochlorothiazide was determined by simultaneous estimation method after eliminating the absorbent of Amlodipine at this wavelength. Validation of the proposed method was carried out for its accuracy, precision and reproducibility according to ICH guidelines. Thus the present study gives an excellent method for the determination of all the 3 drugs in combined dosage formulation without their prior separation [13]. 5. Sharma, Hemendra Kumar; Jain, Nilesh; Jain, Surendra Kumar have documented a new, simple, accurate, precise and reproducible UV spectrophotometric method for the simultaneous estimation of Amlodipine besylate, Olmesartan medoxomil and Hydrochlorthiazide in tablet dosage form. The stock solutions were prepared in methanol. The λmax for Amlodipine besylate, Olmesartan medoxomil and hydrochlorthiazide were nm, nm and nm respectively. The Amlodipine besylate, Olmesartan medoxomil and hydrochlorthiazide obeyed Beer's law in concentration range of 5-25 µg/ml, 6-30 µg/ml and 5-25 µg/ml respectively [14]. 82

11 6. El-Gizawy, Samya M, Abdelmageed, Osama H; Omar, Mahmoud A; Deryea, Sayed M; Abdel-Megied, Ahmed M have devrived a simple, sensitive, and specific method was developed for simultaneous determination of Amlodipine besylate (AML), valsartan (Vals) and Hydrochlorothiazide (HCT) by high performance liquid chromatography without previous separation. Satisfactory resolution was achieved using a RP-C18 chromatographic column, Phenomenex Kinetex (150 mm 4.6 mm i.d) and a mobile phase consisting of acetonitrile-phosphate buffer (0.05 M) with ph 2.8 in the proportion of (40/60, vol./vol.) at a flow rate 0.8 ml/min and the wavelength detection was 227 nm. The method could be used for analysis of combined dose tablet formulation containing AML, Vals, HCT as well as spiked human plasma[15]. 7. Sharma, Hemandra Kumar; Sahu, Vinod; Sahu, Rahul; Sengar, Neha; Kulkerni, Sneha have investigated a simple, precise, rapid, and reproducible RP-HPLC method for the simultaneous determination of Amlodipine besylate (AML) and hydrochlorthiazide (HCZ) present in multicomponent dosage forms. Chromatography was carried out isocratically at 25 ± 0.5 on an Prontosil C18 column ( mm, 5 particle size) with a mobile phase composed of acetonitrile : methanol : phosphate buffer (ph-3) in the ratio of 48:12:40 v/v/v at a flow rate of 1.2 ml/min. Detection was carried out using a UV-PDA detector at 232 nm [16]. 8. Bagyalakshmi, J.; Philip, Sincy Mary; Ravi, T. K. have reported method by using UV spectroscopic by simultaneous equation method. S(-) Amlodipine is a potent calcium channel blocker used for the treatment of hypertension, congestive heart failure and angina pectoris. S(-) Amlodipine avoids the adverse effect of Amlodipine in racemic mixts. Hydrochlorothiazide is a first line diuretic drug of the thiazide class used for the treatment of hypertension, congestive heart failure and symptomatic edema [17]. 9. Sharma, Amrish; Tailang, Mukul; Gupta, Bhaskar; Acharya, Ashish have developed a new and simple reversed phase HPLC method for the determination of Amlodipine in its pharmaceutical dosage form. The mobile phase used acetonitrile, methanol, KH 2 PO 4 was in the ratio of 250:250:500. Buffer solution was prepared by dissolving 0.05 m KH 2 PO % H 3 PO 4 adjunct with triethylamine. The separation was achieved on hypersil C18 column, Phenomenex Gemini ( mm) and 5 µ particle size with rheodyne injector. The flow rate was 1 ml/min and UV detection at 238 nm. The retention time for Amlodipine was 9 min. The linearity coefficient of Amlodipine was 0.99% and the percentage recovery for Amlodipine was % respectively. The 83

12 proposed method was accurate, precise and linear within the desired range. This method can be successfully employed for the quantitative analysis of Amlodipine [18]. 10. Chen, Jing; Yao, Xin-ning; Lin, Li-na; Guo, Xing-jie have investigated the separation of Amlodipine besylate enantiomers using reversed phase high performance liquid chromatography with sulfobutylether-β-cyclodextrin as chiral mobile phase additive. Separation was performed on Hypersil BDS C8 column (150 mm 4.6 mm, 5 µm) with mobile phase composed of water (ph 2.5, containing 25 mmol/l sulfobutylether-β-cyclodextrin)- methanol (v:v = 55:45). The flow rate was 0.8 ml/min and the detection wavelength was set at 238 nm. The effects of some factors such as type and concentration of cyclodextrin and organic modifier, ph of aqueous phase, column temperature and flow rate on the resolution were investigated. Baseline separation of Amlodipine besylate was achieved under the optimized condition (R = 1.6, and α = 1.1). The method is simple and economic with good resolution [19]. 11. Kamble, Asmita Y.; Mahadik, Mahadeo V.; Khatal, Laxman D.; Dhaneshwar, Sunil R. have described two methods for simultaneous determination of Amlodipine besylate and Olmesartan medoxomil in formulation. The first method was based on the HPTLC separation of two drugs on Merck HPTLC aluminum sheets of silica gel 60 F254 using n-butanol: acetic acid: water (5:1:0.1, v/v/v) as the mobile phase. The second method was based on the HPLC separation of the two drugs on the RP-PerfectSil- 100 ODS-3-C18 column from MZ-Analyzetechnik GmbH, Germany and acetonitrile/0.03 M ammonium acetate buffer (ph = 3) in a ratio of 55:45 as the mobile phase. Both methods have been applied to formulation without interference of excipients of formulation [20]. 12. Wei, Xiaoyi; Yang, Gengliang; Qi, Li; Chen, Yi have developed an online solid-phase extraction (SPE)-HPLC method for simultaneous screening of nicardipine and Amlodipine in human plasma. A short monolithic poly (glycidyl methacrylate-coethylene glycol dimethacrylate) [p(gma- EDMA)]-based weak cation-exchange (WCX) column was prepared and employed as the selective extraction sorbent, which exhibited good permeability and biocompatibility. During the online SPE protocol, high-abundance proteins (human serum albumin, IgG, IgA and transferrin) and most matrixes in plasma were fast removed while nicardipine and Amlodipine were effectively trapped on this monolithic column. Furthermore, the monolithic WCX sorbent could be continuously reused more than 300 times without obvious changes in analytes extraction and proteins 84

13 cleanup. Validation assays demonstrated acceptable precision and adequate recovery for simultaneous quantitative screening of nicardipine and Amlodipine in human plasma [21]. 13. Wankhede S B; Wadkar S B; Raka K C; Chitlange S S have documented methods for simultaneous estimation of Amlodipine besilate and Olmesartan medoxomil in pharmaceutical dosage form. Two UV Spectrophotometric and one reverse phase high performance liquid chromatography methods have been developed for the simultaneous estimation of Amlodipine besilate and Olmesartan medoxomil in tablet dosage form. First UV spectrophotometric method was a determination using the simultaneous equation method at nm and nm over the concentration range µg/ml and µg/ml, for Amlodipine besilate and Olmesartan medoxomil with accuracy %, and % respectively. Second UV spectrophotometric method was a determination using the area under curve method at nm and nm over the concentration range of µg/ml and µg/ml, for Amlodipine besilate and Olmesartan medoxomil with accuracy %, and %, respectively. In reverse phase high performance liquid chromatography analysis carried out using 0.05M potassium dihydrogen phosphate buffer : acetonitrile (50:50 v/v) as the mobile phase and Kromasil C18 (4.6 mm i.d.x250 mm) column as the stationery phase with detection wavelength of 238 nm. Flow rate was 1.0 ml/min. Proposed methods can be used for the estimation of Amlodipine besilate and Olmesartan medoxomil in tablet dosage form provided all the validation parameters are met [22]. 14. Rai, Megha; Kawde, P. B. have derived a simple, precise and rapid stabilityindicating HPLC method for the simultaneous quantitative determination of Olmesartan, Amlodipine and Hydrochlorothiazide from their innovative Pharmaceutical combination drug product. The separation was achieved on simple gradient method. The detector wavelength was 260 nm. The described method was validated with respect to system suitability, specificity, linearity, precision and accuracy [23]. 15. Delhiraj, N.; Anbazhagan, S. have developed two new, rapid, precise, accurate and specific chromatographic methods for the simultaneous determination of Olmesartan medoxomil, Amlodipine besylate, and Hydrochlorothiazide in combined pharmaceutical dosage forms. The first method based on reverse phase liquid chromatography by Qualisil BDS C18 column (250 mm 4.6 i.d., 5 µm). Mobile phase consists of 1.0 ml of triethylamine in 1L water and the ph was adjusted to 2.5 with orthophosphoric acid and 85

14 Acetonitrile (60:40) with a flow rate of 1ml/min, with a detection wavelength of 231nm. The second method involved silica gel 60F254 high performance thin layer chromatography and densitometric detection at 231 nm using chloroform: methanol (85:15) as the mobile phase [24]. 16. Doshi, Naman; Sheth, Avani; Patel, C. N. have determined a simple, accurate and precise reverse phase high performance liquid chromatography (RP-HPLC) method for the simultaneous determination of Olmesartan medoxomil, Hydrochlorothiazide and Amlodipine besylate in marketed formulation. The determination was carried out on a Zorbax SB Ph C18 ( mm, 5 µm) column using a mobile phase of sodium perchlorate buffer (ph 3) Buffer solution: 10% THF Containing Acetonitrile (60:40 v/v). The flow rate was 1 ml/min with detection at 250 nm. The results of analysis were validated as per ICH guidelines and by recovery studies [25]. 17. Dubey, Nitin; Jain, Ankit; Raghuwanshi, Ajay K.; Jain, Dinesh K. have investigated a simple, accurate, and precise RP-HPLC method for the simultaneous determination of Olmesartan medoxomil (OLME), Amlodipine besylate (AMLO) and Hydrochlorothiazide (HCTZ) in commercially available tablet formulations. The chromatographic separation was achieved on instrument Shimadzu LC 10 AT VP, Japan, equipped with photodiode array detector (PDA) SPD-10 AVP, attached with a class M 10 A software, (version 1.6) and Phenomenex Luna C8 (25 cm 4.6 mm i.d. 5 µm) column using acetonitrile: phosphate buffer (ph 4 ± 0.1) (40:60 % v/v) as mobile phase at a flow rate of 1.0 ml/min. Quantitation was carried out at 258, 237 and 270 nm for Olmesartan medoxomil, Amlodipine besylate and Hydrochlorothiazide respectively. The method was validated as per ICH guidelines [26]. 18. Jain, P. S.; Patel, M. K.; Gorle, A. P.; Chaudhari, A. J.; Surana, S. J. have documented a simple, specific, accurate, and precise stability-indicating reversed-phase high-performance liquid chromatography method for simultaneous estimation of Olmesartan medoxomil (OLME), Amlodipine besylate (AMLO) and Hydrochlorothiazide (HCTZ) in tablet dosage form. The method was developed using an RP C18 base deactivated silica column ( mm, 5 µm) with a mobile phase consisting of triethylamine (ph 3.0) adjusted with orthophosphoric acid (A) and acetonitrile (B), with a timed gradient program of T/%B: 0/30, 7/70, 8/30, 10/30 with a flow rate of 1.4 ml/min. UV detection was used at 236 nm. The proposed method was validated for precision, accuracy, linearity, range, robustness and ruggedness [27]. 86

15 19. Pawar, A. K. M.; NageswaraRao, A. B. N.; SeshagiriRao, J. V. L. N.; JayathirthaRao, V. have developed and validated a simple isocratic liquid chromatography method for the simultaneous quantitative determination of Hydrochlorothiazide (HCT), Olmesartan medoxomil (OLM) and Amlodipine (AML) in bulk and tablet dosage form. Chromatography was carried on Agilent xbd- RP-18 (5 µm, 150 mm 4.60 mm I.D.) column with mobile phase comprising of acetonitrile, methanol and water (containing 0.1% orthophosphoric acid) in the ratio 25:25:50 v/v and ph was adjusted to 4.6 with sodium hydroxide. The flow rate was adjusted to 0.8 ml/min with UV detection at 232 nm. The different analytical parameters such as linearity, accuracy, precision, robustness, limit of detection (LOD), limit of quantification (LOQ) were determined according to the International Conference on Harmonization (ICH) Q2B guidelines [28]. 20. Rao, Janhavi R.; Rajput, Milindkumar P.; Yadav, Savita S.; Mulla, Toufik S.; Bharekar, Vishal V. have derived a simple, fast, and precise reversed phase, isocratic HPLC method was developed for the separation and quantification of Olmesartan medoxomil, Amlodipine besylate and Hydrochlorothiazide in bulk drug and pharmaceutical dosage form. The quantification was carried out using thermo hypersil ODS-C18 (250 mm 4.6 mm, 5.0 µ) column and mobile phase comprised of methanol:0.05 M potassium dihydrogen phosphate with triethylamine (80:20 v/v) ph 3 adjusted with orthophosphoric acid. The flow rate was 0.8 ml/min and the effluent was monitored at 230 nm. The method was validated in terms of linearity, precision, accuracy, and specificity, limit of detection and limit of quantitation [29]. 21. Manish, Bhatia Neela; Jawaharlal, Deshmane Snehal; Nivrutti, More Harinath; Balkrishna, Choudhari Praffula have developed a simple sensitive, rapid, accurate and precise reverse phase high performance liquid chromatographic method for the simultaneous estimation of Amlodipine besylate and Hydrochlorothiazide from pharmaceutical preparation, blood sample and urine The method involves HIQ Sil C18 5µm column (250 mm 4.6 mm i.d.) using phosphate buffer ph 3.5: Acetonitrile (60:40V/V) as mobile phase at a flow rate of 1ml/min with wavelength detection at 240nm. Losartan potassium was used as an internal standard. The developed method was applied for simultaneous estimation of the two drugs from pharmaceutical formulation, plasma and urine and was validated according to ICH and USFDA guidelines [30]. 87

16 22. Miroshnichenko, I. I.; Lelishentsev, A. A.; Kalmykov, Yu. M.; Fedotov, Yu. A.; Ivashchenko, A. A. have reported a simple and rapid HPLC method with tandem mass spectrometry (TMS) detection has been developed for the determination of Amlodipine in blood plasma. The HPLC system is coupled to a TMS detector via an electrospray ionization (ESI) interface in the positive-ion mode. The plasma samples were extracted either with a mixture of diethylether : hexane (8:2, v/v) by means of solid-phase extraction with Oasis HLB 1-cc cartridges (Waters, USA). The analytes were chromatographed under reversed-phase conditions on a Zorbax 300 SB-C18 column. The proposed HPLC/TMS procedure was successfully used for the study of the clinical pharmacokinetics of Amlodipine and other calcium- channel blockers [31]. 23. Bhatt, Jignesh; Singh, Sadhana; Subbaiah, Gunta; Shah, Bhavin; Kambli, Sandeep; Ameta, Suresh have developed and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the estimation of Amlodipine in human plasma. Amlodipine was extractd from human plasma by using a solid-phase extraction technique. Imipramine was used as the internal standard. A Hypersil BDS C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves a rapid solid-phase extraction from plasma, simple isocratic chromatographic conditions, and mass spectrometric detection that enables detection at sub-nanogram levels [32]. 24. Ustun, F. Gedil; Atay, O. have described IR and HPLC methods for the quantitative determination of Amlodipine (AMP) in a solid dosage form. IR spectroscopic method (KBr disk) was used and disulfiram (DSF) was chosen as the internal standard. The specific absorption bands at 693 cm -1 and 913 cm -1 were chosen for AMP and DSF respectively. In HPLC method good chromatographic separation was achieved using a Luna C18 5 µm ( mm) column and a mobile phase consisting of water-meohphosphate buffer (36:64:1 v/v) at a flow-rate of 0.7 ml/min. Mefrusid (MFD) was chosen as the internal standard. AMP and MFD were detected at 275 nm and eluted at 11.5 and 7.27 min. respectively [33]. 25. BahramiGh; MirzaeeiSh have documented a fast, sensitive and specific high performance liquid chromatographic method using fluorescence detection for analysis of Amlodipine in human serum. Amlodipine is extracted from serum by ethyl acetate and involves precolumn derivatization with 4-chloro-7-nitrobenzofurazan and reverse-phase chromatography on C18 column. The mobile phase was sodium phosphate buffer (ph 88

17 2.5) containing 1 ml/l triethylamine and methanol at flow rate of 2.8 ml/min. Propranolol was used as internal standard. The method has been applied to a bioequivalence study after administration of 10 mg Amlodipine in 12 normal subjects [34]. 26. Baranda, Ana B.; Jimenez, Rosa M.; Alonso, Rosa M. have developed an HPLC method with electrochemical detection for the simultaneous determination of five 1,4-dihydropyridines: amlodipine, nitrendipine, felodipine, lacidipine and lercanidipine. The chromatographic separation was performed on a Supelcosil LC ABZ+Plus C18 column with a mobile phase consisting of MeCN-10 mm acetate buffer (72:28, v/v) at a flow rate of 1 ml/min. The temperature was set at 30 ± 0.2. The amperometric detector, equipped with a glassy C electrode was operated at mv vs. Ag/AgCl in the d.c. mode. The validity of the method was examined comparing the results obtained with those of HPLC with photometric detection [35]. 27. Tatar, S.; Atmaca, S. have developed a sensitive and specific HPLC method using fluorescence detector for the assay of Amlodipine in human plasma. The assay involves derivatization with 4-chloro-7-nitrobenzofurazan (NBD-Cl), solid-phase extraction on a SiO2 column and isocratic reversed- phase chromatography with fluorescence detection. Nortriptyline hydrochloride was used as an internal standard. The plasma profile following a single administration of 10 mg Amlodipine to a healthy volunteer was presented [36]. 28. Feng, Yifan; Meng, Qing; Guo, Xiaoling; Yang, Dongyang; He, Yuan have determined GC-MS extraction ion flow method for the determination of human plasma Amlodipine as an alternative to HPLC. Nitrendipine was used as internal standard, blood samples were made alkaline and a stable, easily evaporable derivative was produced by trifluoroacetyl anhydride derivatization. The detection limit reached 0.35 ng/ml, and satisfied the requirements of determination [37]. 29. Dubey, Nitin; Jain, Ankit; Raghuwanshi, Ajay Kumar; Jain, Dinesh Kumar have reported an HPTLC method for the simultaneous determination of Amlodipine besylate (AMLO), Hydrochlorothiazide (HCTZ), and Olmesartan medoxomil (OLME) in the presence of degrdation products and related impurities in pharmaceutical formulations. Chromatography was performed over precoated silica gel aluminum plates 60 F 254 (10 cm x 10 cm, 200 µm thickness) as stationary phase. Samples were spotted in the form of distinct bands (6 mm in width; 11.6 mm apart) with a CAMAG 100 µl 89

18 syringe using a Linomat V (CAMAG, Switzerland) sample applicator. Equipment parameters were optimized for smooth working (const. application rate 10 µl, scanning speed 100 mm/s; slit dimension 6.00 x 0.45 mm). Linear ascending development was carried out in a 10 cm x 10 cm twin trough glass chamber (CAMAG, Switzerland) previously satd. with optimized mobile phase for 25 min at room temperature (30 C) and relative humidity (RH) of 55 ± 5%. The plates were developed to the distance of 70 mm and dried using hair dryer. The mobile phase (25 ml) was chloroform-et acetate-toluenemethanol-glacial acetic acid (19.5:19.5:38.5: 19.5:3, v/v) with densitometric analysis at 230 nm in absorbance mode with CAMAG TLC Scanner 3 and wincats software. The method was validated in terms of precision, repeatability, accuracy, and other validation parameters. The method could effectively separation the drugs from their degradation products; it may be regarded as stability-indicating [38]. 30. Pandya, K. K.; Satia, Milan; Gandhi, T. P.; Modi, I. A.; Modi, R. I.; Chakravarthy, B. K. have developed novel analytical method for determination of the total plasma levels (free and protein bound) of the calcium channel blocking agent Amlodipine using a high-performance thin-layer chromatography (HPTLC) procedure. Detection and quantitation were performed without internal standards. The present method employs proteolysis of the plasma proteins by incubating plasma for 2 h in pepsin solution. After proteolysis Amlodipine is extracted and a known amount of the extraction is spotted on precoated silica-gel 60 F254 plates using a Camag Linomat IV autosampler. Amlodipine was quantified using a dual-wavelength TLC scanner. The method provides a direct estimation of the total Amlodipine present in plasma [39]. 31. Krishnamurthy, G.; Ahamed, Aleem; Ramesha, S.; Naik, H. S. Bhojya have investigated a novel rapid, sensitive and reproducible, ultra performance liquid chromatography method for quantitative determination of Olmesartan medoxomil in active pharmaceutical ingredients and its dosage forms. Chromatographic separation of drug from the possible impurities and the degradation products was achieved on a Poroshell 120 EC-C mm, 2.7µm column; the gradient elution achieved with in 7.0 min. 0.1% orthophosphoric acid in water and acetonitrile was used as mobile phase. The flow rate was 2.0 ml/min, column temp. 25 C and the detection were done at 210 nm. The above developed UPLC method was further subjected to hydrolytic, oxidative, photolytic and thermal stress conditions. The method was validated for specificity, precision, linearity, accuracy and robustness [40]. 90

19 32. Kumar, Kakumani Kishore; Rao, ChimalakondaKameswara; Madhusudan, G.; Mukkanti, Khagga have described a simple, precise, and rapid stability-indicating ultra-performance liquid chromatography (UPLC) method for the simultaneous quantitative determination of Olmesartan, Amlodipine, and Hydrochlorothiazide from their innovative Pharmaceutical combination drug product, with the presence of degradation products. It involved a 50 mm 2.1 mm, 1.8 µm Ph column. The separation was achieved on simple gradient method. The mobile phase A contains a mixture of sodium perchlorate buffer ph 3.2 (0.053 M): acetonitrile in the ratio 90:10, v/v, and mobile phase B contains a mixture of sodium perchlorate buffer ph 3.2 (0.053 M): acetonitrile in the ratio 10:90, v/v The flow rate was 0.7 ml/min and column temperature was maintained at 55. The gradient program (T/%B) was set as 0/10, 2/50, 4/80, and 6.0/10. The detector wavelength was 271 nm for Hydrochlorothiazide, 215 for Olmesartan and 237 nm for Amlodipine. The described method was validated with respect to system suitability, specificity, linearity, precision and accuracy. The method is fast and is suitable for high-throughput analysis of the drug and one can analyze about 240 samples per working day, facilitating the processing of large-numbers of batch samples [41]. 33. Raj, Shiva; Kumari, K. Siva; Rao, A. Narasimha; Reddy, I. Ugandhar; Raju, M. Naga have highlighted a simple, sensitive, and reproducible ultra-performance liquid chromatography (UPLC) method for the quantitative determination of Olmesartan medoxomil (OLM) in active pharmaceutical ingredient (API) and pharmaceutical dosage forms. Chromatographic separation was achieved on Acquity UPLC BEH Ph 100 mm, 2.1 mm, and 1.7 µm Ph columns and the gradient eluted within a short run time, i.e., within 10.0 min. The eluted compounds were monitored at 210 nm, the flow rate was 0.3 ml/min and the column oven temperature was maintained at 27 C. The drug was subjected to the International Conference on Harmonization (ICH)-prescribed hydrolytic, oxidative, photolytic, and thermal stress conditions. The performance of the method was validated according to the present ICH guide lines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness and robustness [42]. 34. Rahman, Mohammed M.; Khan, SherBahadar; Faisal, M.; Rub, Malik Abdul; Al-Youbi, Abdulrahman O.; Asiri, Abdullah M. have prepared lowdimensional nanoparticles composed SnO2-Co3O4 nanocubes (NCs) by a hydrothermal method using reducing agents. The doped nanomaterials were investigated by UV/visible 91

20 spectroscopy, powder X-ray diffraction, FT-IR, energy- dispersive X-ray spectroscopy (EDS), Raman spectroscopy and field-emission SEM. They were deposited on a silver electrode to give a drug sensor with a fast response towards Olmesartan medoxomil (OSM) in 0.1 M phosphate buffer-phases. The sensor also exhibits higher sensitivity, long-term stability, and enhanced electrochemical response. The sensitivity is µa cm -2 mmol /L and the detection limit is 0.17 nmol /L [43]. 35. Maheshwari, Rajesh Kumar; Rajput, Mithun Singh; Sharma, Satyabrat; Nair, Veena have illustrated the application of mixed hydrotropy. A novel, safe and sensitive method of spectrophotometric estimation in the UV region has been developed using a mixed hydrotropic solution containing 8% each of niacinamide, sodium acetate, sodium benzoate, sodium citrate and urea (total 40% hydrotropic agents), for the quantitative determination of Hydrochlorothiazide, a very slightly water soluble diuretic drug in tablet dosage form. Beer's law was obeyed in the concentration range of µg/ml. There was more than 25-fold enhancement in aqueous solubility of Hydrochlorothiazide in mixed hydrotropic solution as compared with the solubility in distilled water precluding the use of organic solvents. Results of the analysis were validated statistically and by recovery studies. Statistical data proved accuracy, reproducibility and the precision of the proposed method [44]. 36. Zhou, Dan; Liu, Yuxiu; Zhu, Yan have invented ion chromatography-uv detection method for determining content of Hydrochlorothiazide. The method is characterized in that converting Hydrochlorothiazide sample into cationic compound via acid in leaching solution, separating with cation exchange column, and detecting at proper wavelength. The invention is sensitive in separation and detection of Hydrochlorothiazide, and can be used in research of human body fluid pharmacokinetics [45]. 92

21 3. AIM OF PRESENT WORK In recent time, there is increased tendency towards the development of stabilityindicating assays [41-43], using the approach of stress testing as enshrined in the International Conference on Harmonization (ICH) guideline Q1A (R2) [44]. Even this approach is being extended to drug combinations [45, 46] to allow accurate and precise quantitation of multiple drugs in presence of their degradation products and interaction product if any. Various publications are available regarding determination method of Amlodipine besylate, Olmesartan medoxomil and Hydrochlorothiazide, but most of the methods are applicable to alone or combination of two drugs Olmesartan medoxomil, Amlodipine besylate and Hydrochlorothiazide in pharmaceutical dosage form or in biological fluids. Thin-layer chromatography, fluorometric, first-derivative and spectrophotometric, LC- MS and HPLC methods are reported. As far as our knowledge is concern, no stabilityindicating analytical method for the determination of Amlodipine besylate, Olmesartan medoxomil and Hydrochlorothiazide in combine dosage forms has been published. The previous published methods are not directly applicable for this issue and need more investigation for method development and validation. Consequently, the focus in the present study was to develop a validated stability indicating HPLC method for the combination, by degrading the drugs together under various stress conditions like acid hydrolysis, base hydrolysis, oxidation, thermal and photolytic stress which is recommended by ICH guideline. The aim and scope of the proposed work are as under: To develop suitable HPLC method for simultaneous determination Olmesartan medoxomil, Amlodipine besylate and Hydrochlorothiazide in tablet formulation. Forced degradation study of Olmesartan medoxomil, Amlodipine besylate and Hydrochlorothiazide under stress condition. To resolve all major impurities during forced degradation studies of Olmesartan medoxomil, Amlodipine besylate and Hydrochlorothiazide. Perform the validation for the developed method. 93

22 4. EXPERIMENTAL 4.1 Materials Amlodipine besylate and Olmesartan medoxomil standards were provided by Cadila pharmaceuticals Ltd., Ahmadabad (India) and Hydrochlorothiazide standard was provided by Alembic pharmaceuticals Ltd., Baroda (India). Amlodipine besylate, Olmesartan medoxomil and Hydrochlorothiazide tablets containing 10mg Amlodipine besylate, 40mg Olmesartan medoxomil and 25mg Hydrochlorothiazide and the inactive ingredient used in drug matrix were obtained from market. HPLC grade acetonitrile was obtained from Spectrochem Pvt. Ltd., Mumbai (India). Analytical grade ammonium acetate, hydrochloric acid, glacial acetic acid, sodium hydroxide pellets and 30% v/v hydrogen peroxide solution were obtained from Ranbaxy Fine Chemicals, New Delhi (India). 4.2 Instrumentation The chromatographic system used to perform development and validation of this assay method was comprised of a LC-10ATvp binary pump, a SPD-M10Avp photo-diode array detector and a rheodyne manual injector model 7725i with 20μl loop (Shimadzu, Kyoto, Japan) connected to a multi-instrument data acquisition and data processing system (Class-VP 6.13 SP2, Shimadzu). 4.3 Mobile phase preparation The mobile phase consisted of Acetonitrile 0.02M Ammonium acetate buffer ph=4.5 (40:60 v/v). To prepare the buffer solution, g ammonium acetate were weighed and dissolved in 1000 ml HPLC grade water and then adjusted to ph 4.5 with glacial acetic acid. Mobile phase was filtered through a 0.45μm nylon membrane (Millipore Pvt. Ltd. Bangalore, India) and degassed in an ultrasonic bath (Spincotech Pvt. Ltd., Mumbai). 4.4 Diluent preparation Acetonitrile: 0.02M Ammonium acetate buffer (40:60 v/v) is used as diluent. 4.5 Standard preparation Olmesartan medoxomil standard stock solution containing 400µg/ml was prepared in a 100 ml volumetric flask by dissolving mg of Olmesartan medoxomil and then 94

23 diluted to volume with diluent. Further take 25 ml of this stock solution in 50 ml volumetric flask and make up to mark with diluent (this standard solution of 200µg/ml). Amlodipine besylate standard stock solution containing 100µg/ml was prepared in a 100 ml volumetric flask by dissolving mg of Amlodipine besylate and then diluted to volume with diluent. Further take 25 ml of this stock solution in 50 ml volumetric flask and make up to mark with diluent (this standard solution of 50µg/ml). Hydrochlorothiazide standard stock solution containing 250µg/ml was prepared in a 100 ml volumetric flask by dissolving mg of Hydrochlorothiazide and then diluted to volume with diluent. Further take 25 ml of this stock solution in 50 ml volumetric flask and make up to mark with diluent (this standard solution of 125µg/ml). 4.6 Test Preparation Twenty tablets were weighed and the average weight of tablet was determined. From these, five tablets were weighed and transfer into a 500 ml volumetric flask. About 50 ml of diluent was added and sonicated for a minimum 30 minute with intermittent shaking. Then content was brought back to room temperature and diluted to volume with diluent. The sample was filtered through 0.45µm nylon syringe filter..further take 25 ml of this stock solution in 50 ml volumetric flask and make up to mark with diluent. The concentration obtained was 200 µg/ml of Olmesartan medoxomil, 50µg/ml of Amlodipine besylate and 125 µg/ml of Hydrochlorothiazide. 4.7 Chromatographic Conditions Chromatographic analysis was performed on a Phenomenex make Gemini C18 column (250mm 4.6mm i.d., 5 μm particle size) column. The flow rate of the mobile phase was adjusted to 1.0 ml/min and the injection volume was 20 μl. Detection was performed at 241nm. 95

24 5. RESULTS AND DISCUSSION 5.1 Development and Optimization of the HPLC Method In the present work, an analytical method based on LC using UV detection was developed and validated for assay determination of Amlodipine besylate, Olmesartan medoxomil and Hydrochlorothiazide in tablet formulation. The analytical conditions were selected, keeping in mind the different chemical nature of Amlodipine besylate, Olmesartan medoxomil and Hydrochlorothiazide. The development trials were taken by using the degraded sample of each component was done, by keeping them in various extreme conditions. The column selection has been done on the basis of resolution, peak shape, theoretical plates and day-to-day reproducibility of the retention time and resolution between Amlodipine besylate, Olmesartan medoxomil and Hydrochlorothiazide peaks. After evaluating all these factors, Phenomenex make Gemini C18 (250mm 4.6mm i.d., 5 μm particle size) column was found to be giving satisfactory results. The selection of buffer was based on chemical structure of all three drugs. The acidic ph range was found suitable for solubility, resolution, stability, theoretical plates and peak shape of all three components. Best results were obtained with 0.02M ammonium acetate buffer ph 4.5adjusted with glacial acetic acid improved the peak shape of Amlodipine besylate, Olmesartan medoxomil and Hydrochlorothiazide. Finally, by fixing 0.02M ammonium acetate buffer ph 4.5 and mobile phase composition consisting of a mixture of 0.02M ammonium acetate buffer ph Acetonitrile (60:40, v/v). Optimized mobile phase proportion was provided good resolution among all analytes and also from degradation products which were generated during force degradation study. For the selection of organic constituent of mobile phase, acetonitrile was chosen to reduce the longer retention time and to attain good peak shape. Figure 4 represents the wavelength selection graph of standard preparation. Figure 5 and 6 represent the chromatograms of standard and test preparation respectively. 96