Development and Validation of a Novel Second Derivative UV Method for the Estimation of Antihypertensive Drugs in Tablet Dosage Form

Aim: The objective of the current work was to develop and statistically validate a second order derivative method for estimating atenolol in tablet dosage form using UV spectrophotometry. Materials and Methods: The method validation was done in accordance with ICH recommendations. Atenolol (BSC class III) and hydrochloride (BSC class II) were soluble in 0.1 N NaOH and gave stable absorbance with it, hence 0.1 N NaOH was chosen as a solvent. The wavelengths selected were 226 nm and 274 nm for atenolol and hydrochlorothiazide respectively. The proposed method was validated for parameters like linearity, precision, robustness, accuracy, limit of detection and limit of quantification. Results: The method was found to be linear with a correlation coefficient (R 2 ) of 0.999 and within a concentration range of 5-60 µg/mL for atenolol and 5-50 µg/mL for hydrochlorothiazide. The analysis of tablet formulation was carried out using second order derivative method and percentage mean assay was found to be 100.4% and 100.9% for atenolol and hydrochlorothiazide respectively. The method was statistically validated showed less % RSD indicating that method is precise, accurate and robust.


INTRODUCTION
Atenolol (ATEN), a beta-blocker is used to treat angina and high blood pressure.Hydrochlorothiazide (HCTZ) is a thiazide diuretic that increases the urine flow and prevents the retention of fluid in the body.It is used to treathigh blood pressure. 1fferent researchers have employed different methods to estimate these two drugs in combination.8][19][20] Using 0.1 N NaOH as a solvent, the current study aimed to design and validate a second derivative UV method.The method is simple, fast, efficient, rapid and affordable for determining these two drugs in combined dosage form.

Materials
Active Pharmaceutical Ingredients (API) used atenolol (Figure 1) and hydrochlorothiazides (Figure 2) were gifted by Zydus and Unichem Pvt. Ltd., respectively.The formulation ATEN H 25 was purchased from local pharmacy.It contained 50 mg of atenolol and 25 mg of hydrochlorothiazide 0.1 N NaOH was used as solvent for entire study.

Instrumentation and Equipment
UV spectrophotometer of model 1800 and Shimadzu make having UV probe as the software was used.

Choice of solvent
The drug solubility was assessed in a number of solvents like NaOH, ethanol, methanol etc. Depending upon the solubility and the solvent giving stable absorbance reading 0.1 N NaOH was selected for preparation of stock solutions.

Selection of the Wavelength
The selection of wavelengths for the estimation of atenolol and hydrochlorothiazide done by scanning standard solution Zambrekar, et al.: Second Derivative UV Method for Antihypertensives containing 10 µg/mL concentration in the UV region 200-400 nm by using 0.1 N NaOH as blank.The second order derivative spectra were obtained by making use of a derivative mode to examine the overlain spectra.
The amplitude of the second derivative spectrum of atenolol and hydrochlorothiazide was measured at 226.4 nm and 274 nm, respectively from the spectrum.

Preparation of Stock Solutions
A quantity equivalent to 25 mg of atenolol and hydrochlorothiazide was transferred in a 25 ml volumetric flask and dissolved in 0.1 N NaOH to attain a concentration of 1000 µg/mL respectively.Further dilutions were made using this standard stock solution.

Preparation of Calibration Curve
An aliquot of standard solutions of atenolol and hydrochlorothiazide was placed in a set of 10 mL volumetric flasks and the volume was adjusted with solvent, to get concentrations of 5-60 µg/mL and 5-50 µg/mL, respectively for both the drugs.The absorbance of the solution was recorded at a given wavelength and a graph of absorbance vs. concentration was plotted.The regression equation and correlation coefficient (r 2 ) were determined and Beer lambert's range was established.

Method Development and Validation 21
Both the drugs were soluble in 0.1 N NaOH, so this was the solvent chosen for the method development.The method which was developed was then validated as per ICH guidelines

Linearity
The stock solutions were diluted to give a concentration of 5-60 µg/mL and 5-50 µg/mL for atenolol and hydrochlorthiazide to assess the linearity.Calculations of the detection and the quantification limits were based on this.

Precision
The precision of the method was determined by repeatability and intermediate precision.

Repeatability
Repeatability was carried out by analysing the formulation six times for the same concentration.Absorbance of these solutions  was measured at predetermined wavelengths and calculation for the % relative standard deviation was done.

Intermediate Precision
The intermediate precision was checked by intra-day and inter-day analysis.

Intra-day
Intra-day precision was carried out by analysing the tablet formulation of the same concentration and repeating it for three times at different intervals i.e., morning, afternoon and evening on the same day.Absorbance of these solutions measured at predetermined wavelengths and % relative standard deviation was calculated.

Inter-day
Inter-day precision was carried out by the analysing the tablet formulation having same concentration and repeating it three times on three successive days.Absorbance of these solutions measured at predetermined wavelengths and % relative standard deviation was calculated.

Accuracy
Accuracy was checked by performing recovery studies by using pre-analysed sample solution and by evaluating % mean recovery of compounds.The known concentration of drug was added at different concentration level i.e., 80%, 100% and 120%.
In three different 10 mL volumetric flasks 2.5 mL pre-analysed solution was added and 2 mL, 2.5 mL, 3 mL of atenolol standard solution as well as 1 mL, 1.25 mL, 1.5 mL of hydrochlorothiazide standard solution was added and volume was adjusted up to the mark with 0.1 N NaOH.Control was used as blank.Absorbance of solution was recorded at selected wavelengths against blank and the percentage recoveries were calculated.

Robustness
Robustness of developed method was determined by performing the assay procedure.Three minor changes were considered in experimental conditions such as: • Using a different UV spectrophotometric instrument, • Performing analysis by different analyst,

Assay of Formulation
The tablet formulation was powdered and a quantity equivalent to 25 mg of atenolol was appropriately diluted to obtain the required concentration of 12.5 µg/mL of hydrochlorothiazide and 25 µg/ mL of atenolol.The absorbance of these solutions was measured at all selected wavelengths.Drug content and percent purity was calculated.
A set of equations were framed for calculation of concentration of drugs by second order derivative method. 22ter scanning the solutions in between 400 to 200 nm second order derivative spectra were obtained.The concentration of atenolol and hydrochlorothiazide present in tablet were calculated from the regression equation Where   water.According to solubility characteristics, the common solvent for both drugs was found to be 0.1 N NaOH.

Choice of wavelength
Solutions of the working standard of both the drugs were scanned between 200-400 nm against the blank and the spectra of atenolol (Figure 1) and hydrochlorthiazide (Figure 2) were recorded.The choice of wavelength for analysis was made using concept of second order derivative method.
From overlain spectra, two wavelengths were selected: 226.4 nm absorbance due to atenolol only and 274 nm absorbance due to hydrochlorothiazide only.Hence 226.4 nm and 274 nm were selected as wavelengths for analysis which is depicted in Figure 3.

Linearity
Dilutions were made for a linearity range of 5-60 µg/mL for atenolol and 5-50 µg/mL for hydrochlorthiazide.The regression coefficient was found to be in the range (Table 1).
The calibration curve of atenolol and hydrochlorthiazide is displayed in (Figure 4 and Figure 5).

Precision
Analysis was performed by carrying out repeatability studies (Table 2), inter-day and intra-day precision studies (Table 3).
According per the methodology, the solutions were made, and absorbance values were measured.
The precision of the method was confirmed from the % RSD value which was <2% for atenolol and hydrochlorthiazide.

Accuracy
The accuracy of the developed method was confirmed by a recovery study at three different concentrations (80%, 100%, and 120%).
The % recovery at 80%, 100% and 120% for hydrochlorothiazide was found to be 99.85,102.9, and 99.48% respectively and for atenolol was found to be 101.6,99.53, and 98.92% respectively and is within the acceptable range of 95%-105%.The % RSD for atenolol and hydrochlorothiazide was found to be < 2% .Hence, the method developed was accurate (Table 4).

Limit of Detection (LOD) and Limit of Quantification (LOQ)
The slope and standard deviation of the calibration curve were used to determine LOD and LOQ.The developed method was found to be sensitive as seen in Table 5.

Robustness
The typical variations studied under this parameter were UV instrument, analyst and wavelength.As seen above % RSD for atenolol and hydrochlorothiazide for change in UV instrument, analyst and wavelength found to be < 2% .This confirmed the robustness of the developed method (Table 6).

Assay
The above validated method can be employed to estimate atenolol and hydrochlorothiazide in combined dosage form.According to the methodology the solutions were made and absorbance values measured at predetermined wavelengths.The results of atenolol and hydrochlorothiazide were comparable with the corresponding labelled levels of marketed formulation and % assay was found to be 100.9%for hydrochlorothiazide and 100.4% for atenolol which was within the acceptable limit (95%-105%) (Table 7).

DISCUSSION
The above method was developed using 0.    linearity range of hydrochlorothiazide and atenolol were between 5-50 μg/mL and 5-60 μg/mL with correlation coefficient greater than 0.990.The % RSD calculated for precision, robustness and accuracy was found to be < 2% .The percent recoveries results confirm the accuracy of the method.LOD and LOQ study method was performed and observed to be sensitive.The proposed method has been proven to be sensitive, robust, precise and accurate.
The % assay of hydrochlorothiazide and atenolol in tablet formulation gives satisfactory results of 100.8% and 100.6% respectively, which were within the acceptance criteria.

Table 1 : Linearity and range data of atenolol and hydorchlorthiazide.
Atenolol and hydrochlorothiazide were both dissolved separately in different solvents like NaOH, methanol, ethanol and distilled

Table 4 : Accuracy data for atenolol and hydrochlorothiazide. Figure
5: Calibration curve of hydrochlorthiazide.
Zambrekar, et al.: Second Derivative UV Method for Antihypertensives