Rapid ultra-performance liquid chromatography assay of losartan potassium in bulk and formulations
© Amruthraj et al.; licensee Springer. 2014
Received: 16 August 2013
Accepted: 4 April 2014
Published: 28 May 2014
Losartan potassium is a non-peptide AT1 receptor drug used in the treatment of hypertension.
A simple, rapid, sensitive, and validated isocratic reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method was developed and validated for the determination of losartan potassium (LOS) in bulk drug and tablets. The assay was developed using Waters Acquity BEH C18 (100 mm × 2.1 mm), 1.7-μm column with a mobile phase consisting of a mixture of phosphate buffer (pH 3.2) and acetonitrile (50:50 v/v).
An assay with a total run time of only 5 min was developed. The method monitored at 245 nm exhibited linearity over a concentration range of 2.0 to 15.0 μg mL−1 LOS. The limits of detection and quantification (signal-to-noise ratio (S/N) = 10) were found be 0.018 and 0.054 μg mL−1, respectively. The intraday and interday RSDs were less than 1.0%. The method was validated by the determination of LOS levels in tablets where the percentage on the label claim was 100 ± 2. The accuracy of the method was further ascertained by recovery studies via the standard addition procedure, which yielded satisfactory results.
A rapid UPLC assay of LOS in bulk drug and tablets was developed and validated.
The separation science plays a key role in pharmaceutical industry ranging from impurity profiling to the final assay for monitoring the finished products. The batch assay of the pharmaceuticals in a quality control setup needs to be rapid, accurate, and sensitive. The fast quality monitoring of the products using ultra-performance liquid chromatography (UPLC) is gaining pronounced interest. Known for its advanced technology, UPLC is based on the principles of liquid chromatography, which utilizes 1.7-μm column particles. This enhances the separation process without affecting the resolutions. Due to small particle size, the system entails the use of high pressure of the order 15,000 psi to pump the mobile phase. The elevated mobile phase linear velocity results in high resolution, sensitivity, and shorter analysis time. Owing to its speed and sensitivity, this technique is gaining considerable attention in recent years in different fields of pharmaceutical and biomedical analysis (Krishnaiah et al. ; Waren and Tchlitcheff ). The literature survey shows that, despite these advantages, the UPLC has not been applied for the assay of the drug, LOS.
The aim of this study is to develop and validate, according to the current ICH guidelines, a fast, accurate, precise, and sensitive UPLC method for the analysis of LOS in tablets without the interference from inactive ingredients.
Materials and reagents
The pure active ingredient sample of LOS was given as a gift by Jubilant Life Sciences, Mysore, India. Tablet formulations, viz. Cosart from Cipla, and Covance from Ranbaxy, were purchased from local commercial sources. Solvents such as methanol and acetonitrile (HPLC grade), potassium dihydrogen orthophosphate, and orthophosphoric acid (Qualigens, Mumbai, India) were purchased from Merck & Co., Inc., Whitehouse Station, NJ, USA. Double-distilled water was used throughout the investigation.
Chromatographic conditions and equipment
Analyses were carried out on a Waters Acquity UPLC (LabX, Midland, ON, Canada) with a tunable UV detector. The output signal was monitored and processed using an Empower software. The chromatographic column used was Waters Acquity UPLC BEH C18 (100 mm × 2.1 mm with 1.7-μm particle size). The isocratic elution process was adopted throughout the analysis.
Mobile phase preparation
An aqueous phosphate buffer solution was prepared by dissolving 2.000 g of potassium dihydrogen orthophosphate in approximately 400 mL of water. The pH of this solution was adjusted to 3.2 with 10% phosphoric acid before diluting to a final volume of 500.0 mL. A mixture of 500.0 mL phosphate buffer and 500.0 mL acetonitrile was stirred and filtered using a 0.22-μm nylon membrane filter. This solution was also used as the solvent in all the subsequent preparations of analyte samples.
The isocratic flow rate of the mobile phase was maintained at 0.20 mL min−1. The column temperature was adjusted to 40°C. The injection volume was 4.0 μL. The elution was monitored at 220 nm and the total run time was 5.0 min.
Preparation of stock solution
A standard stock solution of LOS (100 μg mL−1) was prepared by dissolving an accurately weighed amount (5.0 mg) of the drug in the solvent to a final volume of 50.0 mL in a volumetric flask.
Procedure for calibration curve
Working standard solutions containing 2.0 to 15.0 μg mL−1 of LOS were prepared by serial dilutions of the stock solution (100 μg mL−1). Aliquots of 4.0 μL were injected (six injections) and eluted with the mobile phase under the reported chromatographic conditions. The average peak area vs. concentration of LOS (in μg mL−1) was plotted to obtain the standard curve for determining the unknown concentrations of the analyte. Alternatively, the corresponding regression equation was derived using mean peak area-concentration data, and the concentration of the unknown analyte can be computed from the regression equation.
Preparation of tablet extracts and assay procedure
Ten tablets (approximately 25-mg LOS each) were weighed and ground to a fine powder. A quantity equivalent to a tablet was weighed and transferred into a 100-mL flask, dissolved in 60 mL of the mobile phase, sonicated for 20 min, and then diluted to the final volume with the same mobile phase to yield a concentration of 250 μg mL−1. The solution was passed through a 0.22-μm nylon membrane filter. Appropriate volumes of the filtered solution were diluted with the mobile phase to obtain the desired concentrations such as 5.0 ppm.
Procedure for method validation
Accuracy and precision
To determine the accuracy and intraday precision, pure LOS solutions of three different concentrations were analyzed in six replicates each during the same day. Mobile phase was injected as the blank solution before the sample injection, and the relative standard deviation (%RSD) values of the peak area and retention time were determined.
Limits of detection and quantification
The limit of detection (LOD) and limit of quantification (LOQ) were determined by the signal-to-noise (S/N) ratio method. These were obtained by a series of dilutions of the LOS stock solution. Precision study was performed at the LOQ level also. LOQ solution was injected six times (n = 6) and the %RSD values for the obtained peak area and retention time were calculated.
Solution and mobile phase stability
The stability of the LOS solution was tested by injecting the sample into the C-18 column. The peak area was recorded at time intervals of 0, 3, 6, 8, 12, and 15 h, and the RSD and time values were calculated. The mobile phase stability was studied by injecting a freshly prepared sample solution at the same time periods, and the RSD values of the peak areas were calculated. The RSD values of both studies were found to be less than 3.0% exhibiting compatibility of the diluent and the stability of the mobile phase.
Results and discussion
The drug LOS is a basic due to the presence of imidazole and tetrazole moieties in the molecule (Figure 1). The log P value of LOS was found to be 2.25 by ChemDraw Ultra Version 7.0 indicating a high lipophilicity. Acidic mobile phase with a buffer of pH 3.2 was chosen to protonate nitrogen atoms for a fast elution. In order to achieve better efficiency of the chromatographic system, the experimental conditions such as composition and pH of the mobile phase, detection wavelength, nature of column, and column temperature were optimized by varying one parameter at a time while keeping the other conditions constant. Several proportions of buffer, water-acetonitrile mixture, and methanol were evaluated to obtain suitable composition of the mobile phase. Parameters such as the retention time, peak shape, theoretical plates, and run time were the major tasks while developing the method. Several combinations of gradient methods were also performed. Isocratic method was found to be better for the assay. Finally, at the mobile phase composition described under the ‘Methods’ section, the method gave the lowest peak tailing factor and the highest theoretical plate count.
Validation of the method
The described method for the assay of LOS has been validated as per the current ICH Q2 (R1) guidelines.
Regression and sensitivity parameters for UPLC analysis of LOS
Linearity range, μg mL−1
2.0 to 15.0
Standard deviation of intercept (S a )
Standard deviation of slope (S b )
Correlation co-efficient (r)
Limit of detection (LOD), μg mL−1
Limit of quantification (LOQ), μg mL−1
Accuracy and precision
Results of determination of LOS in tablets and in standard samples
Tablet brand namea
Nominal amount (mg)
LOS in tablet (μg mL−1)
Pure LOS added (μg mL−1)
Robustness of the method and stability of the solution
Robustness of the analytical method
Column temperature, 15-ppm LOS
−0.097 for 39°C −0.095 for 41°C
0.13 for 39°C 0.12 for 41°C
Wavelength, 15-ppm LOS
243 nm, area
245 nm, area
247 nm, area
−0.093 for 241 nm −0.090 for 245 nm
0.13 for 241 nm 0.06 for 245 nm
Selectivity of the method was evaluated by injecting the mobile phase, standard drug solution, and tablet extract. Under the experimental conditions, there was only a single peak for the drug (LOS) elusion, which showed a high selectivity of the method.
Application to tablet
Aliquots containing 250 μg mL−1 of LOS in extract and the pure drug solution were separately injected in triplicate to the UPLC system. The mean peak area of the tablets was found to be equivalent to that of the pure drug used as a positive control at the same concentration level.
A standard addition procedure was used to evaluate the accuracy and precision of the method. The solutions were prepared by spiking the pure LOS solution into a pre-analyzed LOS tablet extract at three different concentration levels followed by injection into the chromatographic column. The relatively low values of %RE and %RSD show high accuracy and precision of the developed UPLC method for the drug, LOS.
A rapid, isocratic reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method was developed for the quantitative analysis of the drug, losartan potassium or LOS, in pharmaceutical dosage formulations. This method is precise, accurate, robust, and specific. Satisfactory results were obtained from the validation of the method. The short retention time obtained (2.40 min) enables rapid determination of LOS, which is important for its routine analysis in quality control. The method exhibits an excellent performance in terms of sensitivity and speed. Each experiment was repeated three times. Recovery of known amounts of the added analyte was calculated and is presented in Table 2. The reported values of the recovery are the average of three experiments.
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