Comparative study of analytical methods by direct and first-derivative UV spectrophotometry for evaluation of losartan potassium in capsules

Losartan potassium is an antihypertensive non­peptide agent, which exerts its action by specific blockade of angiotensin II receptors. The aim of the present study was the validation and application of analytical methods for the quality control of losartan potassium 50 mg in pharmaceutical capsules, using direct and first­derivative UV spectrophotometry. Based on losartan potassium spectrophotometric characteristics, a signal at 205 nm of the zero­order spectrum and a signal at 234 nm of the first­derivative spectrum, were found adequate for quantification. The results were used to compare these instrumental techniques. The linearity between the signals and concentrations of losartan potassium in the ranges of 3.0­7.0 mg L­1 and 6.0­14.0 mg L­1 for direct and first­derivative spectrophotometry in aqueous solutions, respectively, presented a correlation coefficient (r) of 0.9999 in both cases. The methods were applied for losartan potassium in capsule dosage obtained from local pharmacies, and were shown to be efficient, easy to apply and low cost. These methods do not use polluting reagents and require relatively inexpensive equipment.


INTRODUCTION
Losartan potassium (Figure 1) is an imidazole derivative, chemically described as 2butyl4chloro 1 [p-(o-1H-tetrazol5ylphenyl)benzyl]imidazole5 methanol monopotassium salt.Losartan was developed by DuPontMerck laboratories as a potent nonpeptide angiotensin II receptor (type AT 1 ) antagonist for hyper tension treatment (Barreiro, Fraga, 2001;Korolkovas, 2007).Losartan is administered in its active form and is partially converted into an active metabolite which is responsible for the drug's prolonged pharmacological effect.The therapeutic efficacy of losartan, as well as its renal and antihypertensive effects, seems to be similar to those of angiotensin converting enzyme (ACE) inhibi tors.However, its adverse effects are different, mainly regarding coughing, whose incidence is significantly lower compared with ACE inhibitors (Oigman, 1996).Losartan potassium is a light yellow solid with a mo lecular weight of 461; melting point of 183.5184.5 ºC; aqueous solubility of 3.3 mg mL 1 at pH 7.8 and pKa value of 4.9 (Lastra et al., 2003;Williams et al., 1996).
The literature has described several analytical methods for losartan potassium determination in tablets, when used as a single active principle or in combination with hydrochlorothiazide, using high performance liquid chromatography (HPLC), capillary electrophoresis, super critical fluid chromatography, high performance thin layer chromatography and derivative ultraviolet spectrophotom etry (Ansari et al., 2004;Erk, 2001;Lastra et al., 2003;Mccarthy et al., 1998;Williams et al., 1996).For losartan potassium determination in pharmaceutical capsules, two HPLC methods have been reported (Bonfilio et al., 2009;Maio, Dias, Bergold, 2005).
However, to date, no analytical method has been described in the literature that uses direct or firstderivative spectrophotometry for quantitative analysis of losartan potassium in pharmaceutical capsules, which is fre quently formulated in magistral pharmacies.Moreover, no pharmacopoeia have yet described a monograph for the finished product.
The aim of the present study was to investigate the validation and application of analytical methods for qual ity control of 50 mg losartan potassium pharmaceutical capsules, using both direct and fi rstderivative UV spec direct and firstderivative UV spec trophotometry.
Losartan potassium salt of 98.90� purity sup of 98.90� purity sup sup sup plied by IPCA Laboratories Limited (Athal, India), lot 5069LB2RI and expiration date of 2009/09 was used as the chemical reference substance.Distilled water was used as the solvent.

Losartan potassium stock standard solution
Stock standard solution of losartan potassium was prepared by accurately weighing 25.0 mg of losartan potas sium reference substance into a 50 mL volumetric flask and adding 40 mL of distilled water.The flask was sonicated for 10 min, filled to volume with distilled water, and the solution was then filtered through quantitative filter paper.

Capsule solution
From the average weight obtained of 20 capsules of each product (by subtracting the weight of the shell from the weight of the full 20 capsules), a weight equivalent to 25.0 mg of losartan potassium was accurately transferred Comparative study of analytical methods by direct and firstderivative UV spectrophotometry 149 into a 50 mL volumetric flask to which 40 mL of distilled water was added.The flasks were coded as A, B and C, sonicated for 10 min and filled to volume with distilled water.Subsequently, the solutions were filtered through quantitative filter paper.

Spectrophotometric measurements
Zeroorder spectra were obtained in the UV range 300200 nm with a fixed slit width of 2 nm, scan speed of about 3200 nm min 1 and 0.2 nm data interval.The first derivative spectra were obtained through instrumental electronic differentiation (UVPC version 3.9 software) us ing a delta lambda of 8 nm.The amplitude values, obtained in the firstderivative spectra, were arbitrary units of the distance from the central zero base line to the signal ob tained at 234 nm.The spectrophotometric measurements were recorded using distilled water as a blank solution.

Selectivity
Selectivity was evaluated by zeroorder and the first derivative absorption spectra analyses at 5.0 mg L 1 and 10 mg L 1 , respectively, in the range 300200 nm using losar tan potassium and pharmaceutical capsule products A, B and C solutions.This procedure was also carried out using three excipient mixtures containing the same quantitative composition as the pharmaceutical products A, B and C.

Linearity
For the linearity study, the losartan potassium stock solution was diluted as appropriate with distilled water to obtain final losartan potassium concentrations of 3.0, 4.0, 5.0, 6.0 and 7.0 mg L 1 in five replicates.The analytical curve was produced by plotting drug concentration versus the ab sorbance values at 205 nm.A second analytical curve for the final concentrations of 6.0, 8.0, 10.0, 12.0 and 14.0 mg L 1 was constructed by plotting drug concentration versus amplitude values of the firstderivative spectra at 234 nm.

Precision
Precision was evaluated with respect to both re peatability and intermediate precision.Repeatability was evaluated by analyzing six losartan potassium work standard solution replicates using distilled water as the solvent.The solutions were analyzed using both direct and firstderivative UV spectrophotometry at 5.0 mg L 1 and 10.0 mg L 1 , respectively.This procedure was repeated over a short period of time on the same day.
Intermediate precision was also evaluated by analyz analyz ing six independent losartan potassium solutions, where this procedure was repeated on different days by different analysts.The R.S.D values for determinations were also calculated.

Accuracy
Accuracy was determined by recovery of known amounts of losartan potassium work standard added to the pharmaceutical capsule products A, B and C. In the direct spectrophotometric method, products A, B and C sample solutions were prepared in triplicate at a losartan potassium concentration of 2.5 mg L 1 .Adequate standard solution volumes were added to the sample solutions to obtain final concentrations of 4.0, 5.0 and 6.0 mg L 1 .In the firstderivative spectrophotometric method, products A, B and C sample solutions were prepared in triplicate at a concentration of 5.0 mg L 1 .Adequate standard solution volumes were added to the sample solutions to obtain final concentrations of 8.0, 10.0, and 12.0 mg L 1 .The resulting mixtures were analyzed by the proposed methods and the percentage recoveries were calculated.

Robustness
In order to verify the method's resilience to slight and deliberate variations in the analytical parameters, losartan potassium solutions at 5.0 and 10.0 mg L 1 were analyzed by direct and firstderivative UV spectrophotometric methods, respectively.The robustness was assessed by comparison of the results obtained under standard conditions against those obtained using different cuvettes and at different room temperatures (Turning off the air conditioner).

Detection and quantitation limits
The detection (DL) and quantitation (QL) limits of the spectrophotometric methods were calculated using Equations ( 1) and ( 2 where S.D. is the 20 spectrophotometric blank reading (distilled water) standard deviation and a is the calibration curve slope obtained in the linearity study.

Aqueous stability
The losartan potassium stock solution was diluted to concentrations of 5.0 and 10.0 mg L 1 using distilled water and analyzed by direct and fi rstderivative UV spectropho analyzed by direct and firstderivative UV spectropho tometry, respectively.The analyses were carried out in the periods of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 12 and 24 hours after solution preparation.

Average weight determination
In order to determine the mean weight of pharma pharma ceutical capsule products A, B and C, 20 capsules were weighed and then capsule content removed.The empty shells were weighed, and the content average weight obtained by subtracting the weight of the shell from the weight of the full 20 capsules, as recommended by the Brazilian Pharmacopoeia (F.Bras., 1988).The R.S.D values for determinations were then calculated.

Pharmaceutical capsule assay
The validated analytical methods were applied to determine losartan potassium content for the pharmaceuti pharmaceuti cal capsule products A, B and C using both direct and first derivative UV spectrophotometric methods at 5.0 mg L 1 and 10.0 mg L 1 , respectively.The results were obtained by comparing the sample spectrophotometric measure ments (n=5) with those obtained from losartan potassium standard solutions (n=3) at the same concentration levels.

Content uniformity determination
The content of ten capsules of each product was individually transferred to 100 mL volumetric flasks and 40 mL of distilled water was added.The flasks were sonicated for 10 min and filled to volume with distilled water.The solution was filtered with quantitative filter paper, diluted to concentrations of 5.0 and 10.0 mg L 1 and analyzed by direct and firstderivative UV spectrophoto metric methods, respectively, by comparing the sample spectrophotometric measurements with those obtained from losartan potassium standard solutions (n=3) at the same concentration levels.

RESULTS AND DISCUSSION
Lastra and coworkers studied losartan potassium solution spectrophotometric behavior at various pH values and concluded that a pH variation from 5 to 9 did not alter losartan potassium spectral characteristics (Lastra et al., 2003).Distillation is the most common process used in magistral pharmacies for preparing the purified water to be used in the drugmanipulation and quality control activi ties, and the recommended pH of distilled water is between 5.0 and 7.0 (USP, 2007).The use of water as a solvent in chemical analysis laboratories has many advantages in terms of environmental aspects, cost and safety compared with the use of an organic solvent.Therefore, considering the losartan potassium aqueous solubility of 3.3 mg mL 1 (Lastra et al., 2003;Williams et al., 1996) and the factors mentioned above, the use of distilled water as solvent was deemed adequate for our validation purposes, making the methods simple and convenient.
The zeroorder spectrum of losartan potassium so lution at 5.0 mg L 1 in distilled water showed maximum drug absorption wavelength at 205 nm with absorbance values of 0.5 (Figure 2).Therefore, the absorbance values at 205 nm were used in the direct spectrophotometric me thod while 5.0 mg L 1 was fixed as the 100 percent level on the analytical curve.
Previously published studies justified the use of firstderivative spectrophotometry for losartan potassium determination in tablets by stating that the maximum drug absorption wavelength is close to 205 nm and that no absorption peak is observed at other wavelengths (Lastra et al., 2003).Moreover, the interference from the tablet excipients verified at close to 205 nm, precludes the analytical use of zeroorder spectrophotometry for losartan potassium determination in tablets (Ansari et al., 2004).
The absorption spectrum differentiation through de rivative spectrophotometry does not increase the original spectrum information, but derivative operation reduces the broad band intensity, yielding overlapping peak resolution enhancements, obeying Beer's law (Paschoal et al., 2003).The firstderivative spectrum, obtained through instru mental electronic differentiation of the losartan potassium zeroorder absorption spectrum, shows an intense negative peak at 234 nm (Figure 3).At this wavelength, a losartan potassium solution of 5.0 mg L 1 shows absorbance values of 0.25.For the subsequent analyses, 10.0 mg L 1 was fixed at the 100 percent level of the analytical curve in the first derivative spectrophotometric method in order to produce a analytical signal that corresponded to those observed under the direct spectrophotometric method.
In these experiments, formulation excipients of capsules A, B and C did not present absorbance at 205 nm, demonstrating the direct spectrophotometric method se lectivity for losartan potassium analysis in pharmaceutical capsules (Figure 4).Use of direct spectrophotometry is advantageous because it can be used by analytical labo ratories that do not have access to a spectrophotometer with derivative mode.However, direct spectrophotometric method selectivity was confirmed using only the following excipients: magnesium stearate, aerosil, sodium dodecyl sulfate, talc, starch and microcel MC102 1.Because there is a considerable variety in the excipients used in the drugmanipulation process, the direct spectrophoto metric method selectivity should be further verified for pharmaceutical capsules containing different excipients than those tested in this study, which could be considered a drawback of this method.
The firstderivative spectra analyses showed that capsule A, B and C formulation excipients did not inter did not inter fere in the firstderivative spectrophotometric method (Figure 5).
Although the spectrophotometric methods are not effective for identifying or quantifying impurities and degradation products, the drug substance used in the losartan potassium capsule manipulation should have been adequately analyzed and an analysis certificate should issued by the supplier guaranteeing compliance with the acceptance criteria in accordance with the drug substance monograph on losartan potassium provided by the United States Pharmacopoeia, which establishes a 0.2� limit for any individual impurity and no more than 0.5� of total impurities (USP, 2007).
The linearity results were evaluated by linear regression analysis based on the minimumsquare method.The correlation coefficient value obtained from direct spectrophotometric method over the range 3.0 to 7.0 mg L 1 was r=0.9999 and the calibration equation was: A = 0.095C -0.0048.From the firstderivative spectropho tometry, the correlation coefficient value obtained over the range 6.0 to 14.0 mg L 1 was r=0.9999 and the calibration equation was dA/dl=0.0022C-0.0004.In both cases, the   relative standard deviation of each point (n=5) was lower than 2�.The results show linear correlation between analytical responses and drug concentration.
Overall relative standard deviations for repeatability and intermediate precision were 0.95 � and 0.75 � using direct spectrophotometry.In the case of firstderivative spectrophotometry, the overall relative standard deviations for repeatability and intermediate precision were 0.95 � and 0.75 �.These experimental results confirmed good precision of the methods when performed on the same or different days by different analysts.Relative standard de viations lower than 5� are considered acceptable (Brasil, 2003).
The spectrophotometric analytical methods showed good accuracy, evaluated by the recovery test (Tables I and  II).The mean recovery percentages of 99.00, 101.74 and 98.91 for products A, B and C, respectively, using direct spectrophotometry, and 101.19, 99.86 and 101.56 � for products A, B and C using firstderivative spectrophotome try, corroborated satisfactory recovery.Recovery values from 98.0 to 102.0 � are deemed acceptable.
The robustness test examines the experimental conditions for the method, and the potentially responsible factors such as experimental and environmental condi tions, to be taken into account during method development given that changes in the optimal conditions can result in significant errors.For both analytical methods, the low RSD values demonstrated that the analysis factors (cuvette and room temperature) did not have a significant effect on analytical responses (Tables I and II).
The direct spectrophotometry detection and quan titation limits were found to be 0.01 and 0.04 mg L 1 , respectively, whereas detection and quantitation limits for firstderivative spectrophotometry were found to be 0.07 and 0.23 mg.L 1 , respectively.These results demonstrated that the analyses were being performed in a region above the quantitation limit value.
Aqueous stability of losartan potassium was checked by evaluation of direct and firstderivative spectra at regu lar time intervals up to 24 hours, and yielded no significant alteration in spectral characteristics (Figures 6 and 7).
The average weights of pharmaceutical capsule products A, B and C were 132.33 mg, 155.12 mg and 158.02 mg, respectively.All products showed acceptable weight values as stipulated by the Brazilian Pharmaco poeia, which establishes that capsules of ≤ 300 mg may not deviate by more than 10.0 � w/w from the average weight (F.Bras., 1988).-Aqueous stability evaluation of 5.0 mg L 1 losartan potassium aqueous solutions using zeroorder spectra at regular time intervals up to 24 hours.The methods were applied to losartan potassium pharmaceutical capsule products A, B and C , and the drug percentage values related to label claim are shown in Table III.The results show that the drugs assayed were in accordance with Brazilian Pharmacopoeia, which estab lishes limits between 90 and 110 � of the labeled amount for finished products (F.Bras., 1988).
The content uniformity test showed that individual active content values and relative standard deviations of pharmaceutical capsule products A and C (n=10) were acceptable and met the requirements.Individual values of active content for sample B were found to lie in the range of 85-115�; however the relative standard deviation observed was higher than 6 � (Table III).The individual results should lie between 85 and 115 � of label claim, with a R. S. D. < 6.0 � (F.Bras., 1988).
The proposed analytical methods were compared using individual active content values obtained from the content uniformity test.The statistical analysis was carried out using a 10×3×2 factorial design (ten active content val ues, three products and two methods).Data were submitted to analysis of variance followed by Tukey's test at a 0.05 significance level.The parameters of pharmaceutical cap pharmaceutical cap sule products (A, B and C) and analytical methods (direct and firstderivative spectrophotometry) were compared.The results showed no significant active content values differences among products A, B and C, or between direct and firstderivative spectrophotometric methods (p>0.05).

CONCLUSIONS
Both direct and firstderivative spectrophotometric methods were shown to be efficient, low cost and easy to apply for losartan potassium determination in pharma losartan potassium determination in pharma determination in pharma ceutical capsules.They do not use polluting reagents and require relatively inexpensive equipment.
All validation parameters were found to be highly satisfactory, indicating linearity, selectivity, precision, accuracy, robustness and adequate detection and quan tification limits of both direct and firstderivative spec trophotometric methods.The methods were therefore shown to be suitable for losartan potassium evaluation of pharmaceutical capsules and for routine use in quality control laboratories.

FIGURE 2 -
FIGURE 2 -Losartan potassium zeroorder absorption spectrum at 5 mg L 1 using distilled water as solvent.

FIGURE 3 -
FIGURE 3 -Losartan potassium firstorder absorption spectrum at 5 mg L 1 using distilled water as solvent.

FIGURE 4 -
FIGURE 4 -Zeroorder absorption spectra of standard losartan potassium aqueous solution at 5 mg L 1 , capsule products A, B and C at 5 mg L 1 and A, B and C product excipients.

FIGURE 5 -
FIGURE 5 -Firstderivative absorption spectra of standard losartan potassium aqueous solution at 10 mg L 1 , capsule products A, B and C at 10 mg L 1 and A, B and C product excipients.

FIGURE 7 -
FIGURE 7 -Aqueous stability evaluation of 10.0 mg L 1 losartan potassium aqueous solutions using firstderivative absorption spectra at regular time intervals up to 24 hours.

TABLE I -
Direct spectrophotometry validation values for losartan potassium evaluation in capsules

TABLE III -
Losartan potassium assay and content uniformity test in pharmaceutical capsule products A, B and C