Method optimization and validation for the determination of carvedilol in human blood serum by high-performance liquid chromatography (HPLC)

In this study, the analysis of carvedilol which is an effective third generation beta-blocker in hypertension, congestive heart failure and left ventricular dysfunction following myocardial infarction was investigated in human blood serum by isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method. For the analysis of carvedilol, Supelco Discovery C18 (particle size: 5μm, inner diameter: 4.6 mm and length: 150 mm) reversed phase column as stationary phase and the mixture of acetonitrile : 1% triethylamine (TEA) aqueous solution (pH = 3.50 ± 0.01 adjusted with H3PO4) (30:70 (v/v) mixture) as mobile phase were used. Carvedilol standard solution was analyzed at 210 nm, 240 nm and 254 nm wavelengths by using Diode Array Detector (DAD) and it was observed that carvedilol had a maximum absorbance value at the wavelength of 240 nm. The values of 4, 8 and 16 were tried as bandwidth (bw) and the best results were obtained when bw value was 4. Reference wavelength was determined to be 360 nm. Flow rates of 0.1 mL/min, 0.2 mL/min, 0.3 mL/min, 0.5 mL/min, 1.0 mL/min and 1.5 mL/min were tried as flow rates. The lowest analysis time was obtained at the flow rate of 1.5 mL/min, therefore optimum flow rate was determined to be 1.5 mL/min. Six different temperatures of 25 0C, 30 0C, 35 0C, 40 0C, 45 0C and 50 0C were tried to optimize column temperature. The temperature value of 25 0C was chosen as optimum column temperature because of high column efficiency, reduced peak tailing and high peak area. The volumes of 10 μL, 20 μL, 30 μL, 40 μL and 50 μL were tried as injection volumes and the volume of 40 μL was chosen as optimum injection volume because the injection volume of 50 μL caused a peak fronting. Propranolol, another beta-blocker, was used as internal standard. As a result of analyses, limit of detection (LOD) and limit of quantification (LOQ) were found to be 45.38 ng/mL and 137.50 ng/mL, respectively. The amount of % recovery was determined to be 99.83 ± 0.84. Method was validated by the determination of accuracy, linearity, selectivity, limit of quantification (LOQ), intra-day and inter-day precision, repeatability and short-term and long-term stability parameters.