Simultaneous Analysis of Losartan Potassium, Amlodipine Besylate, and Hydrochlorothiazide in Bulk and in Tablets by High-Performance Thin Layer Chromatography with UV-Absorption Densitometry

A Simple high-performance thin layer chromatography (HPTLC) method for separation and quantitative analysis of losartan potassium, amlodipine, and hydrochlorothiazide in bulk and in pharmaceutical formulations has been established and validated. After extraction with methanol, sample and standard solutions were applied to silica gel plates and developed with chloroform : methanol : acetone : formic acid 7.5 : 1.3 : 0.5 : 0.03 (v/v/v/v) as mobile phase. Zones were scanned densitometrically at 254 nm. The R f values of amlodipine besylate, hydrochlorothiazide, and losartan potassium were 0.35, 0.57, and 0.74, respectively. Calibration plots were linear in the ranges 500–3000 ng per spot for losartan potassium, amlodipine and hydrochlorothiazide, the correlation coefficients, r, were 0.998, 0.998, and 0.999, respectively. The suitability of this method for quantitative determination of these compounds was by validation in accordance with the requirements of pharmaceutical regulatory standards. The method can be used for routine analysis of these drugs in bulk and in formulation.

In recent years TLC has been improved to incorporate HPTLC-grade stationary phases, automated sampleapplication devices, a controlled development environment,

Materials and Reagents.
Analytically pure samples of losartan potassium, hydrochlorothiazide, and amlodipine besylate were procured from Madras Pharmaceuticals, Chennai, as gift samples and used as working standards, methanol of HPLC grade from Merck (Mumbai, India), and chloroform, acetone, and formic acid of analytical reagent grade from S.D. Fine Chemicals were used, without purification to prepare the mobile phase. A solution containing 1 mg/mL losartan potassium, amlodipine besylate, and hydrochlorothiazide was prepared by dissolving 10 mg of each standard in 10 mL methanol and was used as working standard solution.

Sample Preparation. Twenty TRILOPACE * tablets by
Akums Drugs & Pharmaceuticals Ltd containing 50 mg losartan potassium, 5 mg amlodipine besylate, and 12.5 mg hydrochlorothiazide were weighed and powdered. An amount of powder equivalent to 50 mg of LOS, 5 mg of AML, and 12.5 mg of HCZ was transferred to a 50 mL volumetric flask. After addition of 30 mL of methanol and sonication (30 min), the solution was diluted to volume with the same solvent and filtered through a 0.45 µ filter (Millipore, Milford, MA, USA). To this solution known amount of amlodipine standard (20 mg) was added (standard addition method) as its content is very low in the formulation. This solution (1.0, 2.0, and 3.0 µL containing 1000, 2000, and 3000 ng/spot of LOS, 500, 1000, and 1500 ng/spot of AML, and 250, 500, and 750 ng/spot of HCZ) was used for assay of losartan potassium, amlodipine, and hydrochlorothiazide in the tablets.

Chromatography.
Chromatography was performed on 10 cm×10 cm aluminium HPTLC plates coated with 0.2 mm layers of silica gel 60 F 254 (Merck). Samples were applied as 6 mm bands by means of a CAMAG (Muttenz Switzerland) Linomat V automatic sample applicator equipped with a    peak areas of the drugs, taken as a measure of the noise and B is the slope of the corresponding calibration plot. LOQ and LOD for losartan potassium were found to be 0.382 and 0.121 µg/spot, respectively. For amlodipine they were 0.584 and 0.188 µg/spot, respectively, and for hydrochlorothiazide they were 0.497 and 0.162 µg/spot, respectively.

Evaluation of Precision for Assay of the Pharmaceutical
Preparation. The amount of losartan potassium, amlodipine, and hydrochlorothiazide in the pharmaceutical preparation were determined by replicate analysis (n = 3). The results are reported in Table 1. Precision was determined by analysis of standard solutions containing concentrations of LOS, AML, and HCZ covering the entire calibration range. The precision of the method as intraday variation (CV, %) was determined by analysis of these solutions three times on the same day. Interday precision (CV, %) was assessed by analysis of these solutions on three different days over a period of one week. The results of the precision studies are shown in Table 2.

3.2.1.
Accuracy. An accuracy of the method was determined by analysis of standard additions at three different levels, that is, multiple-level recovery studies. The preanalyzed sample solution (2, 0.5, and 0.2 µg/mL of LOS, HCZ, and AML) was spiked with amounts equivalent to 80, 100, and 120% of standard drugs. These solutions were reanalysed, and the recoveries were found to be within the acceptable limits (Table 3).

Specificity.
The mobile phase used was found to be effective in resolving the drugs (Figure 1). The R F values of losartan potassium, amlodipine, and hydrochlorothiazide were 0.74, 0.35, and 0.57, respectively. Typical overlaid absorption spectra of LOS, AML, and HCZ is shown in Figure 2. Peak purity of the drugs was tested by acquiring spectra at the peak start (S), peak apex (A), and peak end can conclude that no impurities or degradation products were coeluted.

Repeatability.
The repeatability of sample preparation was assessed by application of 2 µL standard drug solution six times on a HPTLC plate. After development of plate, peak height and peak area were recorded for the zones. The CV (%) of peak height and area were calculated and found to be 0.45 and 0.56, respectively, for LOS, 0.34 and 0.43 for AML, and 0.67 and 0.32 for HCZ.

Conclusion
The proposed HPTLC method for simultaneous analysis of losartan potassium, amlodipine, and hydrochlorothiazide in pharmaceutical dosage forms has been established for the first time. Use of HPTLC enables analysis of several samples at the same time. The method is very simple, rapid, and provides accurate and precise results.