VC Chauhan*, VN Shah, DA Shah, RR Parmar
Department of Quality Assurance
APMC College of Pharmaceutical Education and Research,
Motipura, Himmatnagar, Gujarat 383001

A  specific,  accurate,  precise  and  reproducible  RP-HPLC  method  has  been  developed  and subsequently validated for the simultaneous determination of Triamterene and Benzthiazide in tablets. The proposed HPLC method utilizes BDS hypersil (Thermo scientific) C18 column (250 mm × 4.6 mm id, 5 μm particle size), and mobile phase consisting of phosphate buffer: methanol (70:30) and pH adjusted to 3.5 with sodium hydroxide and flow rate of 1.0 ml/min. Quantitation was achieved with UV detection at 245 nm based on peak area with linear calibration curves at concentration ranges 10-30 μg/ml for Triamterene and 5-15 μg/ml for Benzthiazide. The retention time of Triamterene and Benzthiazide were found to be 5.960 min and 3.493 min respectively.  The  method  was  validated  in  terms  of  accuracy,  precision,  linearity,  limits  of detection,  limits  of  quantitation  and  robustness.  This  method  has  been  successively  applied  to tablet formulation  and  no interference  from the  formulation excipients  was found.


PharmaTutor (ISSN: 2347 - 7881)

Volume 2, Issue 6

Received On: 17/04/2014; Accepted On: 22/04/2014; Published On: 01/06/2014

How to cite this article: VC Chauhan, VN Shah, DA Shah, RR Parmar; Development and Validation of RP-HPLC Method for Simultaneous Estimation of Triamterene and Benzthiazide in Tablets; PharmaTutor; 2014; 2(6); 115-122

Triamterene is a potassium-sparing diuretic (water pill) that prevents human body from absorbing excessive salt and keeps potassium levels from getting too low. Triamterene is used to treat fluid retention (edema) in individuals with congestive heart failure, cirrhosis of the liver, or a kidney condition called nephrotic syndrome. Triamterene is also used to treat edema caused by having excessive aldosterone in your body[1].

Triamterene chemically is 2,4,7 – triamino, 6- phenylpteridine with a molecular formula C12H11N7 and molecular weight of 253.27 gm/mol[2]. It is an official drug in Indian Pharmacopeia[3].

Figure.1.Chemical structure of Triamterene[2]

Triamterene shows hyperkalemia as its major side effect[4]. So, in order to neutralize this effect it is used in combination with a thiazide diuretic which counteracts the side effect of Triamterene by its hypolkalemic effect[5]. Benzthiazide belong to thiazide class of diuretics, extensively used in treatment of hypertension and edema associated with mild to moderate congestive heart failure. It increases the rate of urine excretion by the kidneys via decreased tubular reabsorption of sodium and chloride ions and by increasing osmotic transport of water to the renal tubules, which in turn decreases cardiac output and blood pressure[6]. On long-lasting thiazide treatment plasma volume and ECF return to normal, but their hypotensive effect continues. This is possibly due to reduced sensitivity of the vascular bed to the circulating catecholamine and angiotensin. Benzthiazide chemically is 6-chloro-3- [ ( phenylmethyl) thio ]methyl ]- 2H- 1,2,4- benzthiadiazine-7-sulfonamide-1,1 dioxide with a molecular formula C15H14ClN3O4S3 and molecular weight of 431.94 gm/mol[7]. Fig.2

Figure.2.Chemical structure of Benzthiazide[2]

Combination of Triamterene and Benzthiazide are used in treatment of edema and hypertension. In the literature survey it was found that Triamterene and Benzthiazide were estimated individually or in combination with other drugs by UV, HPLC, Spectrofluori methods[7-21] and both together estimated by UV spectroscopic method[22]. But no method has been found for simultaneous estimation of Triamterene and Benzthiazide by chromatographic method. In the view of the need in the industry for routine analysis of Triamterene and Benzthiazide in formulation, attempts are being made to develop simple and accurate RP-HPLC method for simultaneous estimation of Triamterene and Benzthiazide and extend it for their determination in formulation.


RP–HPLC instrument equipped with SPD-20 AT UV-Visible detector, (LC-20AT, Shimadzu), Rheodyne injector (20 μl Capacity), BDS hypersil (Thermo scientific) C18 column (250 mm × 4.6 mm, 5 μ particle size) and Spinchrom software was used.

Chemicals and reagents
Reference standard of TRM and BNZ were obtained from Remedix pharma, Bangalore. Methanol used was of HPLC grade and phosphate buffer of (pH 3.5) and all other reagent were of AR grade.

Preparation of standard and test solutions
Preparation of mobile phase

Mobile phase was prepared by mixing of 700 ml of methanol with 300 ml of phosphate buffer, whose pH was previously adjusted to pH 3.5 by addition of sodium hydroxide. The mobile phase prepared was degassed by ultrasonication for 20 min, so as to avoid the disturbances caused by dissolved gases. The degassed mobile phase was filtered through 0.45 μ filters to avoid the column clogging due to smaller particles.

Preparation of standard stock solutions
An accurately weighed quantity of TRM (20 mg) and BNZ (10 mg) were transferred to a 100 ml volumetric flask and dissolved and diluted to the mark with mobile phase to obtain standard solution having concentration of TRM (200 μg/ml) and BNZ (100 μg/ml)

Preparation of solutions for calibrationcurve
The calibration curves were plotted over the concentration range 10-30 μg/ml for TRM and 5-15 μg/ml for BNZ. From the stock solution 200 μg/ml of TRM, the quantity of (0.5 ml, 0.75 ml, 1.0 ml, 1.25 ml, 1.5 ml), and from the stock solution 100 μg/ml of BNZ, the quantity of (0.5 ml, 0.75 ml, 1.0 ml, 1.25 ml, 1.5 ml) were transferred to a series of 10 ml of volumetric flasks and diluted to the mark with mobile phase. Aliquots (20 μl) of each solution were injected under the operating chromatographic conditions described above.

Preparation of sample solution
Take quantity equivalent to 10 mg TRM and 5 mg BNZ was transferred to 100 ml volumetric flask in mobile phase. The solution was filtered through whatman filter paper No. 41 and the volume was adjusted up to the mark with mobile phase. From the above solution 1 ml of solution is taken in 10 ml volumetric flask and suitably diluted with mobile phase to get a final concentration of 10 μg/ml of TRM and 5 μg/ml of BNZ.

The developed method was validated according to ICH guidelines. To check the system performance, the system suitability parameters were measured. System precision was determined on six replicate injections of standard preparations. Number of theoretical plates and asymmetry were measured.

Linearity was performed with five concentrations ranging from 10-30 μg/ml and 5-15 μg/ml for TRM and BNZ respectively. The peak areas versus concentration of drug were plotted and a linear least-square regression analysis was conducted to determine the slope, intercept and correlation coefficient (r) to demonstrate the linearity of the method.

The limit of detection (LOD) and limit ofquantitation (LOQ)
LOD and LOQ of TRM and BNZ were calculated using the following equations as per International Conference on Harmonization (ICH) guidelines.
LOD = 3.3 × σ/S
LOQ = 10 × σ/S
Where σ = the standard deviation of the response
S = Slope of calibration curve.

The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days 3 different concentrations of sample solutions of TRM (10 μg/ml, 20 μg/ml and 30 μg/ml) and BNZ (5 μg/ml, 10 μg/ml and 15 μg/ml). Percentage relative standard deviation (RSD) was calculated

Accuracy was performed by adding known amounts of TRM and BNZ to the pre-analysed tablet formulation and then comparing the added concentration with the found concentration. Three levels of solutions were made which correspond to 80, 100 and 120% of the nominal analytical concentration (10 μg/ml for TRM and 5 μg/ml for BNZ). Each level was prepared in triplicate. The percentage recoveries of TRM and BNZ at each level were determined. The mean recoveries and the relative standard deviation were then calculated.

The robustness of the method was evaluated by assaying the test solutions after slight but deliberate changes in the analytical conditions i.e. flow rate (± 0.2 ml/ min), proportion of buffer and methanol (72:28 and 68:32 v/v), and pH of buffer (± 0.2).



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