Multivariate analytical methods for simultaneous estimation of Atenolol and Hydrochlorothiazide in bulk and tablet dosage form

Instruments and software

Shimadzu FTIR (IR-SPIRIT) is used for the quantification of multi-variate method (CLS method). Shimadzu UV-1800 spectrophotometer with matched pair of 10mm quartz cells is used for UV quantification. Data acquisition was performed by UV Probe software. Instrument was allowed to stand for 10 minutes to stabilize. Then the instrument parameters viz. start wavelength, end wavelength data interval, scan speed, slit width and sample information were entered in the instrument. Then base line correction was performed by keeping the blank in both the sample and reference compartments.

Materials and solvents

Working standards of atenolol and hydrochlorothiazide are procured from Raffles Pharmaceuticals, Tirupati. Methanol was used as the solvent for the current UV study and potassium bromide was used in IR method. Marketed formulation ATEN-H tablets containing 50 mg atenolol and 12.5 mg hydrochlorothiazide manufactured Zydus cadila Pvt. Ltd was the sample selected for the study.

Quantitative analysis by using FTIR spectroscopy

The method employed was classical least squares (CLS) method. In this method, different ratios of the two drugs were prepared and their IR spectrums were recorded between the ranges of 2366.69 – 3433.44cm^-1 and 1564.40 – 1673.30 cm^-1 using FTIR. Total 16 mixtures were prepared with different concentrations of atenolol and hydrochlorothiazide. From these, 10 mixtures were taken as a calibration set and 6 mixtures was taken as a prediction set or validation set.6, 7, 8

Preparation of mixtures

30 mg of atenolol in 300 mg of KBR was taken to get a concentration of 100 µg/ml (stock-1). 30 mg of Hydrochlorothiazide in 300 mg of KBR was taken to get a concentration of 100 µg/ml (stock-1). From this above stock mixtures, different concentrations were prepared to get a series of mixtures.

Calibration of CLS model

The prepared mixtures were taken in a mortar and pestle and triturated with potassium bromide to get a uniform mixture and those mixtures were subjected to hydraulic press to get pellets. The pellets were subjected to FTIR to record the spectra of mixtures. Calibration of CLS model was done by selecting the proper ranges (2366.69 – 3433.44cm^-1 and 1564.40 – 1673.30 cm^-1) using FTIR. By using spectrum values, CLS model was analyzed.

Assay of marketed formulation

Average weight of 20 tablets was taken and made up with KBR up to 200mcg of total weight. The sample was prepared from the mixture to get a concentration of 15mcg of hydrochlorothiazide and 20 mcg of atenolol.

UV Spectrophotometric method development

Different Solvents like Water, Methanol, and 0.1N HCl, 0.1N NaOH and Ethanol were employed for the optimization of the method. Methanol gave a single distinct peak with good absorbance for all the five drugs. So, it was employed as the solvent. From trial-and-error method, λ_max of bulk drugs were determined by preparing the solutions in methanol in UV spectrophotometer and the λ_{max of} atenolol and hydrochlorothiazide was found to be 275.60 nm and 270.4 nm respectively.

Construction of calibration curve

Calibration curve for Atenolol was obtained in the linearity range of 30-180µg/mL by measuring the absorbance at 275.60 nm. Calibration curve for hydrochlorothiazide was obtained in the linearity range of 3-15µg/mL by measuring the absorbance at 270.40 nm. The correlation coefficients for atenolol and hydrochlorothiazide were found to be 0.9996 and 0.9999 respectively.

Application of linear regression analysis method

For linear regression method, two wavelengths (268 nm and 280nm) were considered for the analysis of the component mixture atenolol (ATL) and hydrochlorothiazide (HCTZ). The slope values obtained from the linear regression analysis for each component were used for the formation of the matrix set. The wavelengths selected for the analysis were 268 nm and 280 nm.

The following equation was derived from the linear equation y = mx + c.

The concentrations of the drugs can be calculated by using the following equations.

Amix1-axy1Amix2-axy2=bx1by1bx1by2×CxCyCxCy=bx1by1bx1by2×Amix1-axy1Amix2-axy2

Where:

Percent assay = calculated quantity of test sample(mg)Weight of test sample(mg)×100

Validation of UV spectrophotometric method:

The method was validated for various validation parameters like linearity, range, precision, LOD, LOQ and accuracy as per the ICH guidelines. The validate method was applied to the dosage form to determine the concentrations of atenolol and hydrochlorothiazide.

Figure 1

Spectrum for calibration set of mixtures

Figure 2

Calibration curve for Atenolol

Figure 3

Calibration curve for hydrochlorothiazide

FTIR-CLS quantitative method

FT-IR method like classical least squares (CLS) was developed with algorithm of MLR evolution via K- matrix for mixtures within the range of 2366.69-3433.44; 1564.40-1673.30 cm^-1. The FTIR spectrum of calibration set of mixtures was depicted in Figure 1. The actual and predicted values obtained for the calibration set and validation set mixtures were presented in Table 1, Table 2 respectively. The values obtained were found to be within the range and minimum PRESS values were obtained. Hence this CLS calibration set is optimized and applied to analyze the marketed formulation. The assay values for FTIR- CLS method were found to be 102% and 108 % for Atenolol and Hydrochlorothiazide respectively.

UV Spectrophotometric method

Methanol was selected as solvent keeping solubility, spectral characteristics, cost and environmental safety in view. The UV absorption spectra of the standard solutions of atenolol and hydrochlorothiazide were recorded in the UV region between 200-400 nm and the obtained spectra showed a maximum absorbance at 275.6 nm for atenolol (ATL) and 270.4 nm for Hydrochlorothiazide (HCTZ) respectively. The two wavelengths (λ1 and λ2) considered for ATL (X) and HCTZ (Y) were 275.6 nm and 270.4 nm respectively. The linear regression equations were obtained by using the absorbencies measured at two wavelengths against the concentrations of standard solution for each drug. The calibration curve was constructed in the concentration range of 30-180 µg /mL of ATL and 3-15 µg/mL for HCTZ and the overlay spectra were represented in Figure 2, Figure 3. The linear regression equation was established between concentration and absorbance of various working standard solutions of ATL individually as shown in the Table 3. All the regression parameters showed that a good linear relation exists between the concentration and absorbance. Hence, the method was found to be linear in the range of 30-180 µg/mL for ATL and 3-15µg /mL for HCTZ. The repeatability results showed a % RSD value of less than 2 for both the drugs, which is acceptable and hence the method was precise in terms of repeatability. The assay was calculated from the regression equations that were constructed for both the drugs and the result was found to be 98.5 % (w/w) for ATL and 106% (w/w) for HCTZ. From the overall results for all the analytical performance characteristics, it can be inferred that the developed Linear regression analysis method was simple, precise, accurate and economical.