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Determination of losartan and its degradates in COZAAR® tablets by reversed-phase high-performance thin-layer chromatography

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Determination of losartan and its degradates in COZAAR® tablets by reversed-phase high-performance thin-layer chromatography
  Journal of Pharmaceutical and Biomedical Analysis17 (1998) 671–677 Determination of losartan and its degradates in COZAAR ® tablets by reversed-phase high-performance thin-layerchromatography Kathleen E. McCarthy, Qingxi Wang *, Eric W. Tsai, Rebecca E. Gilbert,Dominic P. Ip, Marvin A. Brooks Pharmaceutical Research and De  elopment ,  Merck Research Laboratories ,  West Point ,  PA  19486  ,  USA Received 26 June 1997; received in revised form 21 October 1997; accepted 21 October 1997 Abstract Losartan potassium is an angiotensin II receptor blocker. It has been formulated and marketed as a tablet dosageform (COZAAR ® ). A reversed-phase high-performance thin-layer chromatography method has been developed forthe determination of losartan and its low level dimeric degradates (E and F). The method has been validated andshown to be sensitive, efficient, and reliable, and can be used as an excellent alternative to the HPLC stability testingof losartan potassium in COZAAR ® tablets. © 1998 Elsevier Science B.V. All rights reserved. Keywords :   HPTLC; Losartan potassium; Quantitation; Stability 1. Introduction Losartan (DuP 753, MK-954), is a potassiumsalt of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2  -(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole(Fig. 1) [1–3]. It has been shown to be an orallyactive, highly specific non-peptide angiotensin IIreceptor blocker. Losartan effectively reduces hy-pertension by suppressing the effects of an-giotensin II at its receptors, thereby blocking therenin-angiotensin system. Losartan has been for-mulated and marketed as COZAAR ® in a tabletdosage form. During the stability study of  Fig. 1. Structure of losartan potassium.* Corresponding author. Tel.:  + 1 215 6521302; fax:  + 1215 6522835; e-mail: qingxi – wang@merck.com0731-7085 / 98 / $19.00 © 1998 Elsevier Science B.V. All rights reserved. PII   S0731-7085(97)00251-3  K  . E  .  McCarthy et al  .  /   J  .  Pharm .  Biomed  .  Anal  .  17 (1998) 671–677  672Fig. 2. Dimeric degradates of losartan potassium in COZAAR ® tablet dosage fomm. COZAAR ® tablets, two diastereoisomeric degra-dates of losartan have been observed, dimers Eand F (Fig. 2). Various analytical methods havebeen developed for the quantitative determinationof losartan in COZAAR ® tablets using analyticaltechniques such as high-performance liquid chro-matography (HPLC) [4], capillary electrophoresis[5], super-fluid chromatography [6], etc. HPLC isthe most widely used technique for the routinestability testing of losartan in COZAAR ® tablets.Thin-layer chromatography (TLC), which isone of the oldest chromatographic methods, iscommonly used in medical-biochemical analysis,food analysis, and environmental pollutant analy-sis [7]. By comparison with HPLC, high-perfor-mance thin-layer chromatography (HPTLC) stillpreserves its advantage as a rapid, reliable andeconomical analytical method. The main applica-tion of TLC in the pharmaceutical industry isintermediate quality control during the develop-ment and production of pharmaceutically activesubstances and the testing of optically pure sub-stances [8–12]. Although TLC is mainly used as adrug screening and confirmation tool [13], quanti-tative pharmaceutical analysis by TLC has re-cently attracted considerable interest due to theimproved technologies with HPTLC. In recentyears, the HPTLC technique has been improvedto incorporate the following features: HPTLC-grade stationary phase, automated sample appli-cation devices, controlled developmentenvironment, automated developing chamber orforce-flow techniques, computer-controlled densit-ometry and quantitation, and fully validated pro-cedures. These features result in methods that arenot only convenient, fast, robust, and cost effi-cient, but also reproducible, accurate and reliable.Applications of HPTLC to the quantitative analy-sis of drug substances in biological and formula-tion matrices have been documented [14]. Forexample, HPTLC methods with densitometric de-tection were developed for the quality control of lidocaine hydrochloride bulk drug and injectionsolution, and the results compared well withHPLC [15].HPTLC methods with stability-indicating fea-tures have been utilized to assess the impurity anddegradation profile of bulk drug substances anddrug formulations [16–20]. By selecting an appro-priate developing solvent and TLC plates, satis-factory separation between active drugs anddegradation products can be achieved. However,  K  . E  .  McCarthy et al  .  /   J  .  Pharm .  Biomed  .  Anal  .  17 (1998) 671–677   673 the sensitivity and accuracy of the methods withregard to low level degradates (0.1% of activedrug) present in the dosage formulation arerarely addressed. This study describes the stabil-ity indicating HPTLC method development andvalidation for the simultaneous determination of losartan and its low level degradates inCOZAAR ® tablet dosage forms. 2. Experimental 2.1.  Materials Losartan potassium and its degradates (dimersE and F) were obtained from Du Pont MerckPharmaceutical Co. (Wilmington, DE, USA).HPLC grade acetonitrile, water, glacial aceticacid, and methanol were obtained from FisherScientific (Fair Lawn, NJ, USA). HPTLC plates(20 cm × 10 cm reversed-phase plates, C18 silicagel on glass) were obtained from Sigma-Aldrich(Milwaukee, WI, USA). 2.2.  Instrumentation and chromatographicconditions The HPTLC system consisted of an automaticTLC sampling device, an automatic developingchamber with programmable solvent flow, heat-ing and cooling control, and a densiometer withfixed slit scanner with wavelength range from200 nm to 800 nm, all from CAMAG Scientific(Wilmington, NC, USA). Data collection anddata analysis were conducted using an on-linecomputer with Cats 3 software (CAMAG Scien-tific).For optimal sensitivity, solutions of the testingsamples were applied to the TLC plates asbands rather than spots. Bands were 6 mm long,and 12   l of sample was applied to each band.The TLC plates were then developed with ace-tonitrile-methanol-0.1% acetic acid (35:25:40, v / v / v) as mobile phase. After developing over adistance of 30 mm, the TLC plates were driedwith hot air for 2 min and with cold air for 4min in the automatic developing chamber. Thedeveloped plates were scanned by linear scan-ning at 4 mm / s with a Scanner II equipped witha PC / AT and Cats 3 software. The scan lengthand width were adjusted to cover the entireband. The analyses were detected with UV at254 nm. 2.3.  Standard solution preparation 2.3.1.  Losartan potassium standard solution A 250.0 mg amount of losartan potassiumwas accurately weighed, dissolved in acetonitrile-water (1:1, v / v) and diluted to volume in a 100ml volumetric flask. Standard solutions were ob-tained by diluting the above stock solution withacetonitrile-water (1:1, v / v) to give 50%, 75%,100%, and 125% levels of the method concentra-tion of 1.0 mg ml -1 . Each solution was thentransferred into an HPTLC vial for analysis. 2.3.2.  Degradate standard solution Two milligram amounts of dimers E and Fauthentic material were accurately weighed andtransferred to a 100 ml volumetric flask. Ace-tonitrile-water (1:1, v / v) was added to volumeand the solution was mixed thoroughly. Thiswas the working standard of dimers E and F.Standard solutions for low level linearity andlimit of detection (LOD) and limit of quantita-tion (LOQ) determination were obtained by di-luting the working standard withacetonitrile-water (1:1, v / v) to concentrationsranging from 0.05% to 2.0% levels of the losar-tan method concentration of 1.0 mg ml -1 . 2.4.  Sample solution preparation One 25 mg COZAAR ® tablet was placed intoeach of ten 25 ml volumetric flasks and dilutedto volume with acetonitrile-water (1:1, v / v). Amagnetic stirring bar was placed into each flaskand stirred for 45 min followed by sonicationfor 10 min. An aliquot was centrifuged and theclear supernatant was transferred into HPTLCvials for analysis.  K  . E  .  McCarthy et al  .  /   J  .  Pharm .  Biomed  .  Anal  .  17 (1998) 671–677  674Fig. 3. Typical chromatogram of COZAAR ® tablets stored at 40°C and 75% relative humidity for 3 years: 1, dimer F; 2, dimer E;3, losartan potassium. (A) COZAAR ® tablet solution; (B) placebo tablet solution. 3. Results and discussion 3.1.  Method de  elopment In this study, the selection of stationary phasewas very important in order to detect low level(0.1% of losartan method concentration of 1.0 mgml -1 ) of dimeric degradates. Normal phase TLCplates were tried during the method development;however, peak tailing was constantly observed forlosartan, and the dimeric degradates, probablydue to the interaction of the tetrazol group of losartan with the silanol group of the TLC plate.This tailing phenomenon reduced the peak sym-metry and the sensitivity of the method dramati-cally. The peak tailing was eliminated by usingC18 reversed-phase HPTLC plates and the sensi-tivity was improved. The selection of mobilephase was also critical to the method sensitivity.Losartan potassium has p K  a  values of 2.36 and5.55. In this respect, using buffer with pH  2.3would be preferred to avoid peak splitting andpeak broadening. Finally, by comparison withspots, bands render sharper peaks and hencehigher sensitivity for the method.Under the conditions described, the  R f   value of losartan standard was found to be 0.79  0.06 andthe  R f   values of dimeric degradates E and F were0.54  0.06 and 0.31  0.06, respectively. Severelystressed COZAAR ® tablets were used to demon-strate the method specificity. A typical chro-matogram of COZAAR ® tablets stressed at 40°C  K  . E  .  McCarthy et al  .  /   J  .  Pharm .  Biomed  .  Anal  .  17 (1998) 671–677   675 and 75% relative humidity for 3 years is shown inFig. 3. Losartan and its dimeric degradates (E andF) are totally baseline separated within 30 mmdeveloping length. Two other peaks with un-known identity were also observed eluting close tolosartan. Since they have not been observed intablets within the shelf-life, these two peaks wereprobably the results of unrealistic stressing of thetablets. Due to the large difference of polarity(hydrophobicity) between losartan and its dimericdegradates, the currently used HPLC method uti-lized a gradient method in order to shorten theanalysis time [4]. In this study, the stationaryphase contains reversed-phase C18 with a largeportion (  40%) of silanol groups which wouldpose stronger interaction with losartan than thedimeric degradates. Overall, the polarity differ-ence between losartan and the dimeric degradatesis compromised on the TLC plate, dramaticallyshortening the elution time. However, by com-parison with HPLC, the measurement repeatabil-ity (precision) and method reproducibility are stilla critical issue with HPTLC methods. Therefore,the instrument parameters and quality of TLCplates have to be well controlled. 3.2.  Method    alidation As demonstrated in Fig. 3, baseline separationof losartan and two dimeric degradates isachieved. Analysis of the sample solution by theHPLC method revealed a similar peak profilewith no extra peaks. There is also no interferenceof either losartan and the degradates from theplacebo, which further demonstrates the specific-ity of this method.The measurement precision of the method wasevaluated by scanning ten spots of the losartanstandard solution. The relative standard deviationof the peak areas was found to be 1.2%.The sensitivity of the method was demonstratedby analyzing dimers E and F at different concen-trations. Three bands of each concentration werescanned and their peak areas were averaged. Thelimit of quantitation of dimers E and F was foundto be 0.1% with a signal / noise ratio of 0.1. Thelimit of detection of dimers E and F was found tobe 0.05% with a signal / noise ratio of greater than Fig. 4. Linearity of detector response versus dimer E degradateconcentrations ranging fro 0.1% to 2% levels of losartanpotassium method concentration (1.0 mg ml − 1 ). 5. A satisfactory linear relationship between thedetector response and concentration of between0.1% and 2.0% of losartan method concentration(1.0 mg ml -1 ) was established for dimers E and F.For example, a correlation coefficient,  R 2 , of 0.995 was obtained with dimer E (Fig. 4).The accuracy of the method was determined byinvestigating the recovery of losartan potassiumat five levels ranging from 50% to 125% of themethod concentration (1.0 mg ml -1 ) from the solu-tion spiked placebo. The results showed goodrecoveries ranging from 99.5% to 100.5% (Table1). The detector responses for losartan solution inthe presence of the placebo tablet were also linearover the range of 50-125% of the method concen-tration (1.0 mg ml -1 ) with a correlation coefficient, R 2 , of 0.994 (Fig. 5). The  y -intercept for thislinear regression is not zero as would be expectedwith HPLC methods. Unlike HPLC methods, for Table 1Accuracy of method determined by the recovery of losartanfrom placebo tablets spiked with losartan potassium solutionsLevel Recovery (%)Amount recov-Amount addedered (mg)(mg)5.0150% 4.98 99.4100.17.5275% 7.5310.0710.02100% 100.599.812.5012.53125%
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