Two Simple and Rapid Spectrophotometric Methods ... - Springer Link

9 downloads 2 Views 161KB Size Report
Olmesartan medoxomil (OLM), (5 methyl 2 oxo. 1,3 dioxolen 4 yl) methoxy 4 (1 hydroxy 1 methyl ethyl) 2 propyl 1 {4 [2 (tetrazol 5 yl) phenyl] phe.

ISSN 10619348, Journal of Analytical Chemistry, 2010, Vol. 65, No. 3, pp. 239–243. © Pleiades Publishing, Ltd., 2010.


Two Simple and Rapid Spectrophotometric Methods for the Determination of a New Antihypertensive Drug Olmesartan in Tablets1 >


Sena C ¸ a g lar, Armag an Önal Department of Analytical Chemistry, Faculty of Pharmacy, University of Istanbul, Beyazit, Istanbul, 34116 Turkey Received August 13, 2008; in final form, June 24, 2009

Abstract—Two simple, rapid and reproducible spectrophotometric methods have been described for the assay of olmesartan (OLM) in pharmaceutical formulation. The methods are based on the formation of ion associates in the reactions between the studied drug substance and ionpair agents [bromocresol green (BCG) and bromophenol blue (BPB)]. By the extraction with dichloromethane and chloroform, yellowcolored ion associates were formed in acidic medium and absorbances were measured at 409 (BCG) and 412 nm (BPB). Optimizations of the reaction conditions were performed. Beer’s law was obeyed within the concentration range from 1–40 μg/mL and 10–120 μg/mL, respectively, for BCG and BPB. The molar absorptivity, detec tion and quantification limits were also determined. The developed methods were applied successfully to the determination of this drug in tablets. DOI: 10.1134/S1061934810030056 1

Olmesartan medoxomil (OLM), (5methyl2oxo 1,3dioxolen4yl) methoxy4(1hydroxy1methyl ethyl)2propyl1{4[2(tetrazol5yl)phenyl] phe nylmethylimidazo5} carboxylate) (Fig. 1) is a potent and selective angiotensin AT1 receptor blocker which has recently been approved for the treatment of hyper tension [1–3]. OLM is also reported to be effective in animal models of atherosclerosis, liver disorders and diabetic nephropathy [4–6]. It is a prodrug and is rap idly deesterified during absorption to form OLM, the active metabolite [7]. OLM was approved by the FDA in April 2002 [8]. To our best knowledge, there is only one UVspectro photometric method and there are no visiblespectro photometric methods reported for the determination of OLM in tablets in literature or pharmacopoeia [9]. The aim of this study is to develop extractive spectro photometric methods for the determination of OLM in drug substance and tablets suitable for routine qual ity control analysis. The method is based on the reac tion of OLM with bromocresol green (BCG) and bro mphenol blue (BPB). Extractive spectrophotometric procedures are popular for their sensitivity in the assay of drugs and, therefore, ionpair extractive spectro photometry has received considerable attention for the quantitative determination of many pharmaceutical compounds [10–13]. The developed methods are sim ple, accurate, precise and sensitive for the determina tion of OLM in tablets.

EXPERIMENTAL Reagents and chemicals. Olmesartan medoxomil (OLM) and Olmetec® tablet (20 mg) were from Pfizer (Istanbul, Turkey). Analyticalreagent grade chemi cals/reagents and bidistilled water were used through out the work. BCG and BPB were obtained from Merck (Darmstadt, Germany). Stock solution of OLM (1 mg/mL) was prepared in acetonitrile and used as standard solution. BCG and BPB solutions of 1.5 × 10–3 M were prepared in 1% ethanol. Phthalate buffer solutions (pH 2.2–5.4) were used for the study. Phthalate buffer solution: 2.042 g of potassium hydrogen phthalate were dissolved in 50 mL of water. The pH was adjusted to the desired value with 0.1 M HCl and the volume was made up to 200 mL with water.

1 The article is published in the original.







Fig. 1. Chemical structure of OLM.









0.6 0.4 0.2


0 300

400 500 Wavelength, nm


Fig. 2. Absorption spectra of the ion associates with OLM (OLMBCG, 40 µg/mL) and (OLMBPB, 15 µg/mL) against reagent blanks.

Apparatus. A Shimadzu UV160A UVvisible spec trophotometer with 1cm matched glass cells was used for the absorbance measurements. pH measurements were made using a WTW pH 526 digital pH meter, cal ibrated with pH 3.0 and 3.5 sodium dihydrogen phos phate standard buffer solution. General procedure. Aliquots of standard solutions (0.01 to 1.2 mL) were transferred into stoppered glass tubes and total volumes were brought to 2 mL with water; 1.2 mL of BCG and 2 mL of BPB reagent solu tions and 1 mL of buffer solutions (for BCG method, pH 3.5 and for BPB method, pH 3) were added in each tube. The reaction mixtures were extracted with 10 mL of organic solvents (dichloromethane for OLMBCG method and chloroform for OLMBPB method were used) for 1 min using a vortex mixer. The two phases were allowed to separate, and the chloroform layers were passed through anhydrous sodium sulphate. The organic layers were transferred into separate 10 mL volumetric flasks, and made up volume with organic solvents. The absorbance of the yellowcolored chlo roform extracts were scanned at 409 nm for BCG method and 412 nm for BPB method against corre sponding reagent blank, prepared similarly except addition of the drug substances. The absorbance (A) was then calculated by the leastsquares method’s regression equation, A = a + bc (where A is the absor bance of 1 cm layer, b is the slope, a is the intercept and c is the concentration of the measured solution in μg/mL). All measurements were performed under ambient laboratory conditions. Procedures for tablets. Five tablets were weighed and powdered. An accurately weighed portion of the powder, equivalent to 100 mg of active ingredient, was transferred into a 100 mL, calibrated volumetric flask. Then a 5 mL portion of solvent was added into the flask containing powdered drug substance. The mix ture was shaken mechanically for five minutes, soni cated in an ultrasonic bath for 30 min, diluted to the

volume with same solvent, mixed, and filtered through a filter. An appropriate aliquot of the filtrate was fur ther diluted with water to the respective volumes to obtain varied concentrations of calibration graphs and assayed as described above (General procedure). Results and discussion. Up to date no visiblespec trophotometric method has been found in literatures or pharmacopoeia for the assay of OLM in tablets. In this study, visiblespectrophotometric methods were for the first time developed to assay OLM in tablets. The proposed methods are simple, rapid and make use of simple reagents, which an ordinary analytical labo ratory can afford. The maximum color development of ion associates formation is completed immediately after all reagents are added. All the measurements were made in 30 min after the preparation of the solutions in all the experiments. No heating or standing was needed. The developed procedures are not time con suming and do not require any expensive equipment. These methods do not involve many procedural steps and do not; take much operator time and expertise like HPLC and other methods. To establish the most convenient conditions, experiments to achieve maximum color intensity in the quantitative determination of the examined drug were carried out. The influence of each of the follow ing variables on the reaction was tested (pH, nature of the solvent, reagent concentration, temperature, shaking time and stoichiometric relationship). The experiments and their results are reported as follows. The drug substance was reacted with BCG and BPB in an acidic buffer to give yellow colored ion associates. It exhibited an absorption maximum at 409 (for OLMBCG) and 412 nm (for OLMBPB) against a reagent blank (Fig. 2). The colorless blanks have practically negligible absorbance. The optimum pH was studied by extracting the col ored ion associates in the presence of phthalate buffers at different pH values (Fig. 3). Optimum pH values for the ion associates are given in Table 1. The effect of solvents such as chloroform, ethyl acetate, ether, dichloromethane, benzene, methyl isobutyl ketone on the color formation was studied. Experimental results indicate that dichloromethane and chloroform were the best solvents for BCG and BPB reactions, respectively. The effect of each of the reagents was studied sepa rately by measuring the absorbances of final solutions resulting from reaction mixtures containing a fixed concentration of OLM and various amounts of the reagent. It was found that a 12fold molar excess of BCG and 20 fold molar excess of BPB were sufficient for the maximum yield of the reactions (Fig. 4). Excess of these dyes did not have any effect either on the color of the ion associate or on the absorbance. The effect of temperature on the colored ion asso ciates was studied at different temperatures. It was found that the colored ion associates were stable up to


Vol. 65

No. 3




0.7 0.6 Absorbance

0.5 0.4 0.3

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0



10 15 20 25 30 35 40 Mole ratio of reagents to olmesartan


Fig. 4. Effect of the reagent concentration on the absor bance of the reaction products (䉬) with BPB, (䊉) with BCG.

0.1 0





pH Fig. 3. Effect of pH on the absorbance of the reaction products (䉱) with BPB, ( ) with BCG.

35°C. Higher temperatures were not suitable due to the volatile nature of dichloromethane and chloro form. The resulting ion associates were found to be stable up to 24 h at room temperature in a dark envi ronment. Using a vortex mixer, shaking intervals ranging from 0.5 to 5 min were studied to determine the most efficient time for ion associate formation. As a conse quence, 1minute shaking time was observed to be the optimum for our study. The stoichiometric ratio of the formed products was investigated by Job’s continuous variation method [14]. A 1 × 10–4 M solution of OLM was used with comparable solutions of the reagent (BCG or BPB). For each method, a series of solutions was prepared in which the total volume of the drugs and reagent was kept at 10 mL, and the procedure was completed as described under General procedures. The application of Job’s continuous variation method indicated 1 : 1 ratio for the drug with each reagent. The calibration graphs were constructed for the two methods under the optimum conditions described above. The molar absorbtivity, concentration range, regression equation and correlation coefficient for each drug is tabulated in Table 1. A linear relationship was found between the absorbance at λmax and the con centration of the drug in the range 1–40 μg/mL for BCG method and 10–120 μg/mL for BPB method in the final measured volume of 10 mL. Regression anal ysis of the Beer’s law plotted at λmax reveals a good cor relation (r2 = 0.9977–0.9988). The graphs showed a negligible intercept, which was calculated by the least squares method’s regression equation. The high molar absorptivities of the resulting colored complexes indi cate high sensitivity of the methods (1.5 × 104–3.9 × 103). The limit of detection (LOD) and quantitation (LOQ) for OLM were determined according to ICH guideline Q2B [15]. LOD was defined as 3 σ/S and JOURNAL OF ANALYTICAL CHEMISTRY

Vol. 65

LOQ was 10 σ/S based on “standard deviation of the response and slope.” The standard deviation of yinter cepts of the regression lines were used as σ (the stan dard deviation of the response); S is the slope of the calibration curve. The results are shown in Table 1. The precision of the proposed methods was investi gated by intraday and interday determinations of OLM at two different concentrations of drug solutions (80 and 100 μg/mL for BPB, 20 and 30 μg/mL for BCG). The intraday studies were performed in one day (for each level n = 5) and interday studies in five days over a period of two weeks. The intra and inter day precision expressed as relative standard deviation values (RSD %) were found to be within 0.88–1.48% and 0.41–1.43%, respectively, for BPB and BCG (Table 2). The data proved good precision for the developed method. To check the accuracy of the proposed method, the standard addition method was applied by adding Table 1. Optical characteristics and statistical data of the re gression equations for the drug reaction with BCG and BPB Parameter


pH 3.5 λmax, nm 409 Beer’s law limita, 1.0–40.0 μg/mL Molar absorptivity, 1.5 × 104 L/mol cm Regression equationb Slope ± SD 0.015 ± 0. 0001 0.2316 ± 0. 0040 Intercept ± SD Correlation coefficient r 2 ± SD 0.9988 ± 0. 0003 LOD, μg/mL 0.05 LOQ, μg/mL 0.17

BPB 3.0 412 10.0–120.0 3.9 × 103

0.0617 ± 0. 00011 0.0059 ± 0. 0015 0.9952 ± 0. 0050 0.8 2.5

a Average of five determinations b A = a + bc (where c is the concentration of drug in µg/mL).

No. 3






Table 2. Intraday and interday precision of olmesartan determination Intraday (n = 5) Actual Concentration, μg/mL Found Concentration, μg/mL BPB


Interday*(n = 5) RSD, %

Found Concentration, μg/mL

RSD, %





















*Results at five different days.

Table 3. Results of recovery studies by standard addition method Amount of drug in tablet, μga Amount of pure drug added, μg BPB

50 50 10 10


30 50 10 20

Total found, μgb (Mean ± S.D.c )

RSD, %

Recovery of pure drug added, %

79.95 99.85 20.19 29.93

0.92 1.05 0.93 1.03

99.83 99.70 101.90 99.65

a Olmetec tablet (20 mg). b Five independent analyses. c Standard deviation.

Table 4. Determination of drugs in dosage forms by the pro posed methodsa Proposed methods Labeled amount, Preparation mg/tablet

Recovery, % BPB




RSD, %


99.23 100.93 0.62 0.53

a Five independent analyses.

known amounts of drugs to the previously analyzed tablet solution. The results are shown in Table 3. The average percent recoveries obtained (101.90–99.65%) indicate good accuracy of the method. The proposed methods were applied to the deter mination of OLM in commercial tablets. The applica bility of the proposed methods for the assay of this drug in tablets was examined and the results are tabulated in Table 4. Five replicate determinations were made. Sat isfactory results obtained for drug are in a good agree ment with the label claims (Table 4). The results were reproducible with low RSD values. The average per cent recoveries obtained are quantitative (99.23% for BPB, 100.93% for BCG), indicating good accuracy of the methods. The results of analysis of the commercial tablets and the recovery study of the drug suggest that

there is no interference from any excipients (such as starch, lactose, titanium dioxide, and magnesium stearate), which are present in tablets. The robustness of the proposed method was evalu ated by using the different instruments by two different analysts under the same optimized conditions. The obtained results were found to be reproducible, since there was no significant difference between the results obtained by the two analysts. Thus, the proposed methods could be considered robust. CONCLUSION Being simple, rapid, sensitive, accurate, and eco nomic, the methods can be recommended for the rou tine determination of OLM in tablets. This is also first report of visiblespectrophotometric determination of OLM in tablets. REFERENCES 1. Mizuno, M., Sada, T., Ikeda, M., Fukuda, N., Miya moto, M., Yanagisawa, H., and Koike, H., Eur. J. Pharmacol., 1995, vol. 285, p. 181. 2. Brunner, H.R., Clin. Ther., 2004, vol 26, p. 28. 3. ChilmanBlair, K. and Rabasseda, X., Drugs Today (Barc), 2003, vol. 39, p. 745. 4. Koike, H., Am. J. Cardiol., 2001, vol. 87, p. 33.


Vol. 65

No. 3


TWO SIMPLE AND RAPID SPECTROPHOTOMETRIC METHODS 5. Kurikawa, N., Suga, M., Kuroda, S., Yamada, K., and Ishikawa, H., Br. J. Pharmacol., 2003, vol. 139, p. 1085. 6. Mizuno, M., Sada, T., Kato, M., and Koike, H., Hypertens. Res., 2002, vol. 25, p. 271. 7. Yoshida, K. and Kohzuki, M., Cardiovasc. Drug Rev., 2004, vol. 22, p. 285. 8. Electronic Resource, tent.nsf/news/8525697700573E1885256D40004A2A4C. 9. Celebier, M., Altünoz, S., Pharmazie, 2007, vol. 62, p. 419. 10. Barary, M.H. and Wahbi, A.M., Drug Dev. Ind. Pharm., 1991, vol. 17, p. 457.


Vol. 65


11. Abdelmageed O.H. and Khasaba, P.Y., Talanta, 1993, vol. 40, p. 1289. 12. Botello, J.C. and PerezCaballero, G., Talanta, 1995, vol. 42, p. 105. 13. Sastry, C.S.P., Rama Rao, K., and Siva Prasad, D., Tal anta, 1995, vol. 42, p. 311. 14. Miller, J.C. and Miller, J.N., Statistics for Analytical Chemistry, Chichester: Ellis Horwood, 1993, 3rd ed. 15. International Conference on Harmonization. Note for Guidance on Validation of Analytical Procedures: Meth odology, Switzerland: Committee for Proprietary Med ical Products, 1996.

No. 3


Suggest Documents