Derivative and Q-analysis Spectrophotometric Methods for Estimation of Hydrochlorothiazide and Olmesartan Medoxomil in Tablets

Two methods for simultaneous estimation of hydrochlorothiazide and olmesartan medoxomil in combined tablet dosage form have been developed. The first method is the application of Q–analysis method (absorbance ratio), which involves the formation of Q–absorbance equation at 264 nm (isobestic point) and at 271 nm, the maximum absorption of hydrochlorothiazide. The linearity ranges for hydrochlorothiazide and olmesartan medoxomil were 2.5-22.5 μg/ml and 4-36 μg/ml, respectively. The second method is based on the derivative spectrophotometric method at zero crossing wavelengths. The linearity ranges for hydrochlorothiazide and olmesartan medoxomil were 2.5-20 μg/ml and 4-32 μg/ml, respectively. The accuracy of the methods were assessed by recovery studies and was found to be 100.45% ±0.4215 and 100.24% ±0.3783 for absorbance ratio method and 99.39% ±0.221 and 99.72% ±0.11 for first derivative method, for hydrochlorothiazide and olmesartan medoxomil, respectively. These methods are simple, accurate and rapid, those require no preliminary separation and can therefore be used for routine analysis of both drugs in quality control laboratories.

HPTLC method has been reported for its estimation in combination with HCTZ [22,23] .The dose of OLME is 20 mg daily and its structure is shown in fi g. 2. A combination of drugs, HCTZ (12.5 mg) and OLME (20 mg) in tablet formulation is available commercially (Olmezest-H 20, Sun Pharmaceutical Industries Ltd., Mumbai, India).However, no spectrophotometric method has yet been reported for simultaneous estimation of HCTZ and OLME.Hence, an attempt has been made to develop and validate, in accordance with ICH guidelines, a simple, precise, accurate and economical spectrophotometric method for quantitative analysis of HCTZ and OLME in combined tablets [24,25] .

MATERIALS AND METHODS
Pharmaceutically pure sample of HCTZ and OLME were obtained as generous gifts from Golden Cross Pvt. Ltd., Daman, India and MSN Laboratories Pvt. Ltd., Medak, India, respectively.Methanol AR grade (Merck Ltd., Mumbai, India) was used as solvent in the study.Double beam UV/Vis spectrophotometer, Shimadzu model 1601 with a pair of 10 mm matched quartz cells was used to measure absorbance of the resulting solution.

Preparation of standard stock solution:
Accurately, 50 mg each of HCTZ and OLME was weighed separately and transferred to two different 50 ml volumetric fl ask.Each drug was dissolved in methanol and volume was made up to the mark with methanol.The standard stock solution (1000 µg/ml) were further diluted separately to obtain working standard solution of concentration 7.5 µg/ml of HCTZ and 12.0 µg/ml of OLME.

Study of spectra and selection of wavelengths:
Each working standard solution was scanned between the range 200-400 nm in 1 cm cell against blank.The zero and fi rst order derivative absorption spectra were recorded.Two wavelengths were selected from the overlain zero order spectra (fi g. 3), 264 nm (Isobestic point) and 271 nm (λ max of HCTZ) for formation of Q-absorbance equation.The peak amplitude of fi rst derivative spectra (fi g. 4) was measured at 254.5 nm and 269.5 nm for HCTZ and OLME, respectively.

Procedure for analysis of tablet formulation:
Twenty tablets were accurately weighed and average weight was calculated.The tablets were triturated to a fine powder.An accurately weighed quantity of powder equivalent to 60 mg of OLME was dissolved in methanol and volume was made up to 50 ml.The solution was filtered through Whatmann filter paper No. 41 and aliquot portion of filtrate was diluted to produce solution of 7.5 μg/ml of HCTZ and 12 μg/ml of OLME.The absorbance of sample solution was measured at selected wavelengths and the concentrations of the two drugs were estimated using absorbance ratio and first order derivative methods.The analysis procedure was repeated six times and the results are depicted in Table 1.

RESULTS AND DISCUSSION
In quantitative estimation of two components by Q-analysis method, absorbances were measured at the isobestic wavelength and maximum absorption wavelength of one of the two drugs.From overlain spectra of HCTZ and OLME (fig.3), absorbances were measured at the selected wavelengths i.e., 264 nm (isobestic point) and at 271 nm, the maximum absorption of HCTZ.The absorptivity coeffi cients of each drug at both wavelengths were determined.The concentration of each drug in laboratory mixture and tablet formulation was determined by substituting the absorbance and absorptivity coefficients in the following sets of equations, Cx= Qm-Qy/Qx-Qy×A/ Ax 1 (Eqn.1) and Cy= Qm-Qx/Qy-Qx×A/Ay 1 (Eqn.2), where, Cx is the concentration of HCTZ, Cy is the concentration of OLME, Qm is the ratio of absorbance of sample at selected wavelengths, Qx is the ratio of absorptivity coeffi cients of HCTZ, Qy is the ratio of absorptivity coeffi cients of OLME, Ax 1 is absorptivity coeffi cient of HCTZ at 264 nm, Ay 1 is absorptivity coeffi cient of OLME at 264 nm.
Upon examining the first derivative spectra of the two drugs (fig.4), it can be noticed that HCTZ can be determined at 254.5 nm where OLME has no contribution and OLME can be determined at 269.5 nm where HCTZ shows a zero crossing.The concentrations of drugs were determined from the standard calibration curve of HCTZ and OLME, respectively by interpolation method.A= 0.0028c+0.0009,r= 0.9977 (λ= 254.5nm) (Eqn.3) and A= 0.0016c+0.0002,r= 0.9998 (λ= 269.5nm) (Eqn.4), where, c is the concentration in µg/ml, A is the peak amplitude of the fi rst derivative curves at 254.5 nm and 269.5 nm for HCTZ and OLME respectively, r is correlation coeffi cient.The peak amplitude of fi rst derivative spectra was measured at 254.5 nm and 26.5 nm for HCTZ and OLME, respectively.The amount of the two drugs was calculated from the computed regression Eqns. 3 and 4. The results are depicted in Table 1.
The methods were validated with respect to linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and ruggedness.To study accuracy of the developed methods, recovery studies were carried out using standard addition method at three different levels.Percent recovery and low relative standard deviation value shows accuracy of the spectrophotometric methods.For precision of methods, the relative standard deviation for six replicates of sample solution was less than 2%, which met the acceptance criteria established for spectrophotometric methods.Ruggedness of the proposed method was determined by analysis

Fig. 4 :
Fig. 4: Overlain fi rst order derivative spectra of hydrochlorothiazide and olmesartan medoxomil I is hydrochlorothiazide; II is olmesartan medoxomil; III and IV are zero crossing points of hydrochlorothiazide (269.5 nm) and olmesartan medoxomil (254.5 nm), respectively.

TABLE 3 : VALIDATION PARAMETERS
LOD is limit of detection; LOQ is limit of quantifi cation; % RSD is percentage relative standard deviation.HCTZ is hydrochlorothiazide and OLME is olmesartan medoxomil.