# Development and validation of HPTLC and spectrophotometric methods for simultaneous determination of candesartan cilexetil and hydrochlorothiazide in pharmaceutical preparation.

Introduction

Candesartan cilexetil (CAN) is one of the angiotensin-II-receptor antagonists which are safe and effective agents for the treatment of hypertension and heart failure, either alone, or in conjunction with hydrochlorothiazide (HYD), a thiazide diuretic[1]. They selectively block the angiotensin type 1 (AT1) receptor, which is responsible for vasoconstriction, and they prevent salt and water retention while the angiotensin type 2 (AT2) receptor, which is thought to have cardioprotective effects is not affected[2, 3]. Thiazide diuretics are used in the treatment of oedema associated with mild to moderate congestive heart failure and in the control of hypertension, either alone, or in combination with other drugs, such as angiotensin-II-receptor antagonists. Their hypotensive effect is believed to initially arise from the reduction of blood volume by sodium depletion, and later on, by direct relaxation of arteriolar smooth muscle[1].

CAN is a non-official drug. Few methods have been reported for its determination in pharmaceuticals including HPLC[4] and voltammetry[5]. In biological fluids, it has been determined almost exclusively based on HPLC[6-9]. In the BP[10], HYD bulk powder is assayed by non-aqueous titration with 0.1M tetrabutylammonium hydroxide and the end point is determined potentiometrically, while HYD tablets are assayed spectrophotometrically at [[lambda].sub.max] = 273 nm. On the other hand, the USP[11] describes an HPLC method for the assay of HYD either in bulk powder or in tablets. Several methods have been reported for the assay of HYD. In pharmaceutical formulations, it has been assayed using differential pulse anodic voltammetry[12], HPLC[13-16], TLC[17, 18] and spectrophotometry[19-21]. In human plasma and urine, it has been determined using several HPLC[22-24] methods.

The literature revealed few methods for the simultaneous determination of CAN and HYD in pharmaceuticals including HPLC [25, 26], derivative and derivative ratio spectrophotometry [27] and capillary electrophoresis [28]. To our knowledge no HPTLC or difference spectrophotomertic methods, for the determination of CAN/HYD mixture in pharmaceuticals, have been reported in the literature to date. Therefore, the aim of the present work is the development and validation of a reliable HPTLC and simple selective difference spectrophotometric methods for the simultaneous determination of CAN and HYD in their combined tablet formulation. Both methods were validated in compliance with ICH guidelines[29].

Experimental

Instrumentation

HPTLC plates (20 x10 cm, aluminum plates with 250 um thickness precoated with silica gel 60 [F.sub.254]) were purchased from E. Merck (Darmstadt, Germany). The samples were applied to the plates using a 100 [micro]l Camag microsyringe (Hamilton, Bonaduz, Switzerland) in the form of bands using a Camag Linomat IV (Switzerland) applicator. The slit dimension was kept at 6 x 0.2 mm and 20 mm [s.sup.-1] scanning speed was employed. Ascending development of the mobile phase was carried out in 20 cm x 10cm twin trough glass chamber (Camag, Switzerland). Densitometric scanning was performed at 240 nm on a Camag TLC scanner III operated in the reflectance-absorbance mode and controlled by CATS software (V 3.15, Camag). The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm.

Spectrophotometric measurements were performed using Perkin-Elmer Lambda EZ201 UV-visible spectrophotometer with matched 1-cm quartz cells. The instrument is connected to Panasonic impact dot matrix printer KX-P3626.

Materials and reagents

Pharmaceutical grades of CAN and HYD were kindly supplied by (Pharonia Pharmaceuticals, New Borg El-Arab City, Alexandria, A.R.E,) and (Pharco Pharmaceuticals, Alexandria, Egypt), respectively, and certified to contain 99.98 and 99.90 %, respectively. Methanol, chloroform, hydrochloric acid (BDH Laboratory Suppliers, Poole, England) and sodium hydroxide (El-Nasr Chemical Ind. Co.,Egypt) were analytical grade.

The commercial Atacand-Plus[R] tablets (Batch No. HA4474) manufactured by Astra Zenica and labeled to contain 12.5 mg HYD and 16 mg CAN/tablet were used.

HPTLC conditions

The HPTLC plates were developed with chloroform: methanol (8:2, v/v) as mobile phase. For detection and quantification 10 [micro]l of test and standard solution within the linearity range (Table 1) were applied as separate compact bands of 6 mm width, 6 mm apart and 15 mm from the bottom of the plate. The plate was developed up to the top (over a distance of 8 cm) in the usual ascending way. The chromatographic tank was saturated with mobile phase in the usual manner. After elution, the plate was air dried and scanned at 270 nm, as under the described instrumental parameters.

Standard solutions

Stock standard solutions of 100 mg% CAN or HYD were prepared in methanol. The standard solutions were appropriately diluted to prepare the working solutions for the proposed methods.

Construction of calibration curves

HPTLC method

Standard solutions were freshly prepared by dilution of the stock solution with methanol to obtain final concentrations of CAN and HYD as cited in table 1. Ten microliters of each standard solution were applied to the HPTLC plate. The plate was developed using the previously described mobile phase. The peak areas were plotted against the corresponding concentrations to obtain the calibration graph.

Difference and derivative-difference spectrophotometric methods

Appropriate volumes of the stock standard solution of each drug (to give final concentration within the linearity ranges mentioned in table 1) were separately transferred into two sets of 10-ml calibrated volumetric flasks, then the volume was completed to 4 ml with methanol. One set was diluted to volume with 0.1 M NaOH and the other set was diluted to volume with 0.1 M HCl. The spectra of each drug in 0.06 M methanolic NaOH were recorded against the corresponding solutions of the drugs in 0.06 M methanolic HCl as a blank. The absolute amplitudes of [DELTA]A at 292 nm and 338 nm were plotted versus the concentrations of CAN and HYD, respectively. The absolute values of the [DELTA][D.sup.2] amplitudes were measured at 296 nm (zero-crossing of HYD) and [DELTA][D.sup.1] amplitudes at 299 nm (zero-crossing of CAN) were measured and found to be proportional to the concentration of CAN and HYD, respectively.

Analysis of tablets

A total of 10 Atacand-Plus[R] tablets were accurately weighed and finely powdered. An accurate weight of the powder equivalent to 25 mg HYD and 32 mg CAN was transferred into a 25-ml volumetric flask using about 15 ml methanol and shaken for 30 min., the solution was then completed to volume with methanol, and filtered. The procedure was completed on the filtrate as previously mentioned in sections 2.5.1. and 2.5.2.

Results and discussion

HPTLC method

The experimental conditions for HPTLC method such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible compact, flat, dark fluorescence quenched bands against a bright green background for the simultaneous determination of CAN and HYD. A scanning wavelength of 270 nm was chosen as a common wavelength to match the concentration ratio of the drugs present in their formulation[30].

The greatest differences between the RF values of the two compounds (0.46 for HYD and 0.74 for CAN), with minimum tailing were obtained by using a mobile phase consisting of chloroform: methanol (8:2, v/v). Fig. 1 shows that the two compounds could be separated with good resolution with sharp and symmetrical peaks.

[FIGURE 1 OMITTED]

Difference spectrophotometric method

Fig. 2. shows the zero order spectra of CAN (48 [micro]g [ml.sup.-1]) and HYD (37.5 [micro]g [ml.sup.-1]) in 0.06 M methanolic HCl and NaOH. The difference absorption spectra of CAN, HYD and their mixture are shown in fig.3a. CAN can be successfully estimated by measuring its [DELTA]A value at 292 nm which corresponds to a zero-crossing for HYD whereas HYD can be determined by measuring its [DELTA]A value at 338 nm which corresponds to a zero-crossing for CAN. Figures 3b and 3c. show the first and second derivative difference spectra of both drugs and their mixture (the influence of [DELTA][lambda] on the first and second derivative of the difference spectra was tested and [DELTA][lambda] = 6 nm was considered suitable for both drugs). In fig. 3b HYD shows a well defined maximum at 299 nm, while CAN has a zero [DELTA][D.sup.1] value at the same wavelength. On the other hand in fig. 3c CAN has a [DELTA][D.sup.2] at 296 nm while HYD exhibits no contribution.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Validation

ICH guidelines [29] for method validation were followed for the developed HPTLC, [DELTA]A and [DELTA]D methods. All validation parameters are shown in tables 1, 2 and 3.

Linearity

The linearity of the proposed methods was evaluated by analyzing series of different concentrations of each of CAN and HYD. According to ICH, at least five concentrations must be used.

Under the experimental conditions described, the graphs obtained by plotting peak area (for HPTLC), [DELTA]A, [DELTA][D.sup.1] and [DELTA][D.sup.2] values versus concentration (in the ranges stated in table 1) show linear relationships. The slopes, intercepts and correlation coefficients obtained by the linear least squares regression treatment of the results are also given. An important statistical parameter for indicating the random error in the estimated values of y is the standard error of the estimate, or the standard deviation about regression, or the standard deviation of the residuals, [S.sub.y/x]. The smaller the standard error of the estimate the closer the points are to the straight line. Standard deviation of intercept ([S.sub.a]) and of slope ([S.sub.b]) are also presented for each compound using the proposed methods of measurements. The high values of the correlation coefficients (r-values > 0.999) with negligible intercepts together with the high F-values indicate the good linearity of the calibration graphs[31, 32]. The linearity was further evaluated by calculation of the percentage relative SD of the slope ([S.sub.b]%). Also, the small degree of scatter of the experimental data point around the line of regressions was confirmed by the small values of the variances around the slopes [S.sub.b.sup.2]. For more confirmation, the Student's t-test was performed to determine whether the experimental intercept (a) of the above-mentioned regression lines was not significantly different from the null hypothesis. The calculated values of t (a/[S.sub.a]) do not exceed the 95% criterion of t = 2.31 for 5 samples. So the intercepts are not significantly different from zero in the proposed methods. Thus, the hypothesis that (a) is of negligible value is confirmed[31, 32].

For equal degrees of freedom, increase in the variance ratio (F-values) means increase in the mean of squares due to regression and decrease in the mean of squares due to residuals. The greater the mean of squares due to regression, the more the steepness of the regression line is. The smaller the mean of squares due to residuals, the less the scatter of the experimental points around the regression line. Consequently, regression lines with high F-values (low significance F) are much better than those with lower ones. Good regression lines show high values for both (r) and (F) values[32].

Limit of Detection and Limit of Quantitation

For HPTLC, limit of detection (LOD) is considered as the concentration which has a signal-to-noise ratio of 3:1. For limit of quantitation (LOQ), the ratio considered was 10:1 with a RSD% value less than 10%[29]. For spectrophotometric methods, LOD and LOQ were calculated using the formulae given by Miller[31] where the limit of detection, LOD = 3 S/b and the limit of quantitation, LOQ = 10 S/b, where S is the standard deviation of replicate blank responses (under the same conditions as for sample analysis) and b is the sensitivity, namely the slope of the calibration graph. Using the proposed methods, LOD and LOQ for each compound were calculated and are presented in table 1.

Accuracy

The accuracy of the proposed methods was evaluated by analyzing five laboratory-prepared mixtures of CAN and HYD at various concentration ratios within the working range of each compound (Table 2). Satisfactory recoveries, small relative errors, with small relative standard deviations (RSD%) were obtained, which indicated the high accuracy of the proposed methods.

Precision

The intra-day and inter-day variation for the determination of CAN and HYD were carried out at three different concentration levels namely; 0.15, 0.50, 0.70 [micro]g x [band.sup.-1] (CAN) and 0.05, 0.25, 0.50 [micro]g.[band.sup.-1] (HYD) for HPTLC. Meanwhile, 20, 60, 100 [micro]g [ml.sup.-1] (CAN) and 20, 50.0, 70.0 [micro]g [ml.sup.-1] (HYD) were used for evaluating the precision of spectrophotometric methods. Method repeatability was obtained from RSD% values obtained by repeating the assay five times on the same day for intra-day precision (Table 3). Intermediate precision was assessed by the assay of the sample sets on three different days (inter-day precision). The RSD% values depicted in Table 3 shows that proposed HPTLC and spectrophotometric methods provides acceptable intra-day and inter-day variation of CAN and HYD.

Robustness

Robustness of the proposed methods was evaluated by analyzing CAN and HYD at the same concentration levels mentioned in 3.3.4 for precision. For HPTLC method, the parameters studied were mobile phase composition, mobile phase volume, duration of saturation, time from chromatography to scanning and different plates. However for spectrophotometric methods, the parameters included, molarity of HCl and NaOH, methanol:water ratio used for preparing methanolic HCl and NaOH solutions, different models of spectrophotometers and different lots of solvent. It was found that variation in the above parameters had no significant influence on the determination of CAN and HYD using the proposed methods. The low values of RSD% of peak areas along with nearly unchanged retardation factor ([R.sub.F]) (for HPTLC method) and the low RSD% of [DELTA]A, [DELTA][D.sup.1] and [DELTA][D.sup.2] values (for spectrophotometric methods) obtained after introducing small deliberate changes in the method parameters indicated the robustness of the developed methods (Table 3).

Selectivity

The selectivity was checked by analyzing synthetic mixtures containing different ratios of both drugs, where good percentage recoveries were obtained indicating that they did not interfere with each other (Table 2). In addition, the application of the proposed method for the determination of the two drugs in dosage forms; without interference from the excipients clearly demonstrate the selectivity of the method (Table 4).

Stability in solutions

The stability of CAN and HYD in their solutions during the analytical procedures was studied. Solutions of the two drugs were prepared and stored at room temperature for 0.5, 1.0, 2 hrs. They were then analyzed using the proposed methods since no additional peaks were found in the chromatograms throughout the analysis time using HPTLC, this indicates the stability of both drugs in the sample solution. Using the spectrophotometric methods, no significant changes in [DELTA]A, [DELTA][D.sup.1] and [DELTA][D.sup.2] values were obtained throughout the analysis time, thus the two drugs are stable in solutions for at least 2 hours.

Furthermore, for HPTLC, Spot stability is very important. The time which the sample is left to stand prior to chromatographic development can influence the stability of separated spots and are required to be investigated for validation [33]. Two-dimensional chromatography using the same solvent system was used to find out any decomposition occurring during spotting and development. In case, if decomposition occurs during development, peak(s) of decomposition product(s) shall be obtained for the analyte both in the first and second direction of the run. Since no decomposition was observed during spotting and development using the proposed conditions, this indicated the stability of drugs in solutions.

Analysis of tablets

The proposed methods were applied to the determination of CAN and HYD in commercial tablets. Five replicate determinations were made. Satisfactory results were obtained for both drugs and were in good agreement with the label claims (Table 4). Moreover, to check the validity of the proposed methods, the standard addition method was applied by adding CAN and HYD to the previously analyzed tablets. The recovery of each drug was calculated by comparing the concentration obtained from the spiked mixtures with those of the pure drug. The results of analysis of the commercial tablets and the recovery study (standard addition method) of both drugs (Table 4) suggested that there is no interference from any tablet excipients, which are present in tablets. The results of determination of CAN and HYD in tablets obtained by the proposed methods were compared with those of the reference spectrophotometric method [27]. A statistical comparison of the results was performed with regard to accuracy and precision using Student's t-test and the F-ratio at a 95% confidence level (Table 4). There is no significant difference between the proposed methods and the reference method with regard to accuracy and precision.

Conclusion

The proposed HPTLC, difference and derivative difference spectrophotometric methods provide simple, accurate, reproducible and specified quantitative analysis for simultaneous determination of candesartan cilexetil and hydrochlorothiazide in tablets. The application of difference spectrophotometry is very advantageous in quantitative and qualitative analysis. The main goal of this method is to increase the selectivity of the measurement by eliminating the effect of foreign light absorption, if the spectra of these substances do not change with the use of different pH values. Moreover, application of the derivative technique to difference spectrophotometry adds to the sensitivity of the method. The HPTLC method has some advantages such as a short run time, large sample capacity and minimal volume use of solvent. With these two methods, one can gain the advantages of speed, lower cost, and environmental protection without sacrificing accuracy.

References

[1] G.H. Cocolas, J.N. Delgado, W.A. Remers, 1998, Textbook of Organic Medicinal and Pharmaceutical Chemistry, tenth ed., Lippincott Raven, Philadelphia, New York.

[2] T. Unger, 1999,"Significance of angiotensin type 1 receptor blockade: why are angiotensin II receptor blockers different?," Am. J. Cardiol., 84, pp. 9S-15S.

[3] R. Willenheimer, B. Dahlof, E. Rydberg, L. Erhardt, 1999,"AT1-receptor blockers in hypertension and heart failure: clinical experience and future directions," Eur. Heart J., 20, pp. 997-1008.

[4] D.V.S. Rao, P. Radhakrishnanand, M.V. Suryanarayan, 2007,"A stability-indicating LC method for candesartan cilexetil," Chromatographia, 66, pp. 499-507.

[5] B. Dogan, B. Uslu, S.A. Ozkan, 2004,"Anodic adsorptive stripping voltammetry of the antihypertensive drug candesartan cilexetil at a glassy carbon electrode.," Die Pharmazie, 59, pp. 840-844.

[6] J.Q. Yuan, S.M. Li, H. Wang, J. Yu, W.T. Jing, 2006,"RP-HPLC determination of candesartan in human plasma with fluorometric detection," Yaowu Fenxi Zazhi, 26, pp. 761-763.

[7] L. Gonzalez, J.A. Lopez, R.M. Alonso, R.M. Jimenez, 2002,"Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography with fluorimetric detection.," J. Chromatogr., 949, pp. 49-60.

[8] H. Stenhoff, P.O. Lagerstrom, C. Andersen, 1999,"Determination of candesartan cilexetil, candesartan and a metabolite in human plasma and urine by liquid chromatography and fluorimetric detection.," J. Chromatogr. B:Biomed. Appl., 731, pp. 411-417.

[9] J.W. Lee, N.D. Weng, T. Johnson, A. Dzerk, T. Miyabayashi, M. Motohashi, 1995,"Development and validation of column-switching high-performance liquid-chromatographic methods for the determination of a potent AII receptor antagonist, TCV 116, and its metabolites in human serum and urine," J. Chromatogr. B:Biomed. Appl., 670, pp. 287-298.

[10] The British Pharmacopeia (2007), Her Majesty's Stationery Office, London.

[11] The United States Pharmacopeia, Twenty-ninth edition, The National Formulary, Twenty-fourth edition (2007), United States Pharmacopeial Convention, Inc.

[12] O. Abdel-Razak, 2004,"Electrochemical study of hydrochlorothiazide and its determination in urine and tablets " J. Pharm. Biomed. Anal., 34, pp. 433-440.

[13] L.R. Bhat, R.K. Godge, A.T. Vora, M.C. Damle, 2007,"Validated RP-HPLC method for simultaneous determination of telmisartan and hydrochlorothiazide in pharmaceutical formulation," J. Liq. Chrromatogr. Rel. Tech., 30, pp. 3059-3067.

[14] Y.Q. Wei, H.B. Xou, Y.J. R., 2006,"Simultaneous LC determination of chlorogenic acid and hydrochlorthiazide in Zhenju Jiangya tablets," Chromatographia, 64, pp. 709-711.

[15] S. Erturk, S.M. Centin, S. Atmaca, 2003,"Simultaneous determination of moexipril hydrochloride and hydrochlorothiazide in tablets by derivative spectrophotometric and high-performance liquid chromatographic methods," J. Pharm. Biomed. Anal., 33, pp. 505-511.

[16] E. Dinc, O. Ustundag, 2005,"Application of multivariate calibration techniques to HPLC data for quantitative analysis of a binary mixture of hydrochlorathiazide and losartan in tablets," Chromatographia, 61, pp. 237-244.

[17] D. Kowalczuk, H. Hopkala, R.J. Pietras, 2003,"Simultaneous densitometric determination of quinapril and hydrochlorothiazide in the combination tablets," J. Planar Chromatogr. Mod. TLC, 16, pp. 196-200.

[18] S.A. Shah, I.S. Rathod, B.N. Suhagia, S.S. Savale, J.B. Patel, 2001,"Simultaneous determination of losartan and hydrochlorothiazide in combined dosage forms by first-derivative spectroscopy and high-performance thin-layer chromatography," J.AOACInt., 84, pp. 1715-1723.

[19] D. Kowalczuk, H. Hopkala, 2004,"Application of derivative spectrophotometry for simultaneous determination of quinapril and hydrochlorothiazide in the combination tablets," J.AOAC Int., 87, pp. 847-851.

[20] E. Dinc, B. Uslu, S.A. Ozkan, 2004,"Spectral Resolution Of A Binary Mixture Containing Valsartan And Hydrochlorothiazide In Tablets By Ratio Spectra Derivative And Invers Least Square Techniques," Anal. Lett., 37, pp. 679-693.

[21] N. Erk, 2003,"Three new spectrophotometric methods applied to the simultaneous determination of hydrochlorothiazide and irbesartan," Die Pharmazie, 58, pp. 543-548.

[22] T.M. Huang, Z. He, B. Yang, L.P. Shao, X.W. Zheng, G.L. Duan, 2006,"Simultaneous determination of captopril and hydrochlorothiazide in human plasma by reverse-phase HPLC from linear gradient elution.," J. Pharm. Biomed. Anal., 41, pp. 644-648.

[23] A. Vonaparti, M. Kazanis, I. Panderi, 2006,"Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the determination of benazepril, benazeprilat and hydrochlorothiazide in human plasma," J. Mass Spect., 41, pp. 593-605.

[24] A.A. El-Emam, S.H. Hansen, M.A. Moustafa, S.M. El-Ashry, D.T. El Sherbiny, 2004,"Determination of lisinopril in dosage forms and spiked human plasma through derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) followed by spectrophotometry or HPLC with fluorimetric detection," J. Pharm. Biomed. Anal., 34, pp. 35-44.

[25] N. Erk, 2003,"Simultaneous Analysis of Candesartan Cilexetil and Hydrochlorothiazide in Human Plasma and Dosage Forms Using HPLC with a Photodiode Array Detector " J. Liq. Chrromatogr. Rel. Tech., 26, pp. 2581-2591.

[26] J. Lei, X.S. Zhang, Z. Zhuo, K. Zhu, P. Sun, Q. Fan, 2007,"HPLC-UV simultaneous determination of candesartan cilexetil and hydrochlorothiazide in compound candesartan cilexetil tablets," Yaowu Fenxi Zazhi, 27, pp. 566-568.

[27] N. Erk, 2003,"Application of first derivative UV-spectrophotometry and ratio derivative spectrophotometry for the simultaneous determination of candesartan cilexetil and hydrochlorothiazide.," Die Pharmazie, 58, pp. 796-800.

[28] S. Hillaert, W. Van Den Bossche, 2003,"Simultaneous determination of Hydrochlorothiazide and several angiotensin-II-receptor antagonists by capillary electrophoresis," J. Pharm. Biomed. Anal., 31, pp. 329-339.

[29] I. Q2A, Q2A (R1), Validation of Analytical Procedures: Text and Methodology, International Conference on Harmonization, Geneva, (November 2005), (http://ww.ich.org/LOB/media/MEDIA417.pdf),

[30] A.C. Moffat, M.D. Osselton, B. Widdop, 2004, Clarke's Analysis of Drugs and Poisons, thirrd edition, London.

[31] J.N. Miller, J.C. Miller, 2000, Statistics and Chemometrics for Analytical Chemistry, 4th ed., Prentice Hall, Harlow, England.

[32] P. Armitage, G. Berry, 1994, Statistical Methods in Medical Research, 3 rd ed., Blackwell Scientific Publications, Oxford, England.

[33] P.D. Sethi, 1996, High Performance Thin Layer Chromatography: Quantitative Analysis of Pharmaceutical Formulations, CBS Publishers and Distributors, New Delhi, India.

Rasha M. Youssef (a) *, Hadir M. Mahera, Ekram M. Hassan (a,b), Eman I. El-Kimary (a) and Magda A. Barary (a)

(a) Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, University of Alexandria, El-Messalah, Alexandria 21521, Egypt

(b) Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, Beirut Arab University

E-mail: rmmy1973@yahoo.com

Candesartan cilexetil (CAN) is one of the angiotensin-II-receptor antagonists which are safe and effective agents for the treatment of hypertension and heart failure, either alone, or in conjunction with hydrochlorothiazide (HYD), a thiazide diuretic[1]. They selectively block the angiotensin type 1 (AT1) receptor, which is responsible for vasoconstriction, and they prevent salt and water retention while the angiotensin type 2 (AT2) receptor, which is thought to have cardioprotective effects is not affected[2, 3]. Thiazide diuretics are used in the treatment of oedema associated with mild to moderate congestive heart failure and in the control of hypertension, either alone, or in combination with other drugs, such as angiotensin-II-receptor antagonists. Their hypotensive effect is believed to initially arise from the reduction of blood volume by sodium depletion, and later on, by direct relaxation of arteriolar smooth muscle[1].

CAN is a non-official drug. Few methods have been reported for its determination in pharmaceuticals including HPLC[4] and voltammetry[5]. In biological fluids, it has been determined almost exclusively based on HPLC[6-9]. In the BP[10], HYD bulk powder is assayed by non-aqueous titration with 0.1M tetrabutylammonium hydroxide and the end point is determined potentiometrically, while HYD tablets are assayed spectrophotometrically at [[lambda].sub.max] = 273 nm. On the other hand, the USP[11] describes an HPLC method for the assay of HYD either in bulk powder or in tablets. Several methods have been reported for the assay of HYD. In pharmaceutical formulations, it has been assayed using differential pulse anodic voltammetry[12], HPLC[13-16], TLC[17, 18] and spectrophotometry[19-21]. In human plasma and urine, it has been determined using several HPLC[22-24] methods.

The literature revealed few methods for the simultaneous determination of CAN and HYD in pharmaceuticals including HPLC [25, 26], derivative and derivative ratio spectrophotometry [27] and capillary electrophoresis [28]. To our knowledge no HPTLC or difference spectrophotomertic methods, for the determination of CAN/HYD mixture in pharmaceuticals, have been reported in the literature to date. Therefore, the aim of the present work is the development and validation of a reliable HPTLC and simple selective difference spectrophotometric methods for the simultaneous determination of CAN and HYD in their combined tablet formulation. Both methods were validated in compliance with ICH guidelines[29].

Experimental

Instrumentation

HPTLC plates (20 x10 cm, aluminum plates with 250 um thickness precoated with silica gel 60 [F.sub.254]) were purchased from E. Merck (Darmstadt, Germany). The samples were applied to the plates using a 100 [micro]l Camag microsyringe (Hamilton, Bonaduz, Switzerland) in the form of bands using a Camag Linomat IV (Switzerland) applicator. The slit dimension was kept at 6 x 0.2 mm and 20 mm [s.sup.-1] scanning speed was employed. Ascending development of the mobile phase was carried out in 20 cm x 10cm twin trough glass chamber (Camag, Switzerland). Densitometric scanning was performed at 240 nm on a Camag TLC scanner III operated in the reflectance-absorbance mode and controlled by CATS software (V 3.15, Camag). The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 190 and 400 nm.

Spectrophotometric measurements were performed using Perkin-Elmer Lambda EZ201 UV-visible spectrophotometer with matched 1-cm quartz cells. The instrument is connected to Panasonic impact dot matrix printer KX-P3626.

Materials and reagents

Pharmaceutical grades of CAN and HYD were kindly supplied by (Pharonia Pharmaceuticals, New Borg El-Arab City, Alexandria, A.R.E,) and (Pharco Pharmaceuticals, Alexandria, Egypt), respectively, and certified to contain 99.98 and 99.90 %, respectively. Methanol, chloroform, hydrochloric acid (BDH Laboratory Suppliers, Poole, England) and sodium hydroxide (El-Nasr Chemical Ind. Co.,Egypt) were analytical grade.

The commercial Atacand-Plus[R] tablets (Batch No. HA4474) manufactured by Astra Zenica and labeled to contain 12.5 mg HYD and 16 mg CAN/tablet were used.

HPTLC conditions

The HPTLC plates were developed with chloroform: methanol (8:2, v/v) as mobile phase. For detection and quantification 10 [micro]l of test and standard solution within the linearity range (Table 1) were applied as separate compact bands of 6 mm width, 6 mm apart and 15 mm from the bottom of the plate. The plate was developed up to the top (over a distance of 8 cm) in the usual ascending way. The chromatographic tank was saturated with mobile phase in the usual manner. After elution, the plate was air dried and scanned at 270 nm, as under the described instrumental parameters.

Standard solutions

Stock standard solutions of 100 mg% CAN or HYD were prepared in methanol. The standard solutions were appropriately diluted to prepare the working solutions for the proposed methods.

Construction of calibration curves

HPTLC method

Standard solutions were freshly prepared by dilution of the stock solution with methanol to obtain final concentrations of CAN and HYD as cited in table 1. Ten microliters of each standard solution were applied to the HPTLC plate. The plate was developed using the previously described mobile phase. The peak areas were plotted against the corresponding concentrations to obtain the calibration graph.

Difference and derivative-difference spectrophotometric methods

Appropriate volumes of the stock standard solution of each drug (to give final concentration within the linearity ranges mentioned in table 1) were separately transferred into two sets of 10-ml calibrated volumetric flasks, then the volume was completed to 4 ml with methanol. One set was diluted to volume with 0.1 M NaOH and the other set was diluted to volume with 0.1 M HCl. The spectra of each drug in 0.06 M methanolic NaOH were recorded against the corresponding solutions of the drugs in 0.06 M methanolic HCl as a blank. The absolute amplitudes of [DELTA]A at 292 nm and 338 nm were plotted versus the concentrations of CAN and HYD, respectively. The absolute values of the [DELTA][D.sup.2] amplitudes were measured at 296 nm (zero-crossing of HYD) and [DELTA][D.sup.1] amplitudes at 299 nm (zero-crossing of CAN) were measured and found to be proportional to the concentration of CAN and HYD, respectively.

Analysis of tablets

A total of 10 Atacand-Plus[R] tablets were accurately weighed and finely powdered. An accurate weight of the powder equivalent to 25 mg HYD and 32 mg CAN was transferred into a 25-ml volumetric flask using about 15 ml methanol and shaken for 30 min., the solution was then completed to volume with methanol, and filtered. The procedure was completed on the filtrate as previously mentioned in sections 2.5.1. and 2.5.2.

Results and discussion

HPTLC method

The experimental conditions for HPTLC method such as mobile phase composition and wavelength of detection were optimized to provide accurate, precise and reproducible compact, flat, dark fluorescence quenched bands against a bright green background for the simultaneous determination of CAN and HYD. A scanning wavelength of 270 nm was chosen as a common wavelength to match the concentration ratio of the drugs present in their formulation[30].

The greatest differences between the RF values of the two compounds (0.46 for HYD and 0.74 for CAN), with minimum tailing were obtained by using a mobile phase consisting of chloroform: methanol (8:2, v/v). Fig. 1 shows that the two compounds could be separated with good resolution with sharp and symmetrical peaks.

[FIGURE 1 OMITTED]

Difference spectrophotometric method

Fig. 2. shows the zero order spectra of CAN (48 [micro]g [ml.sup.-1]) and HYD (37.5 [micro]g [ml.sup.-1]) in 0.06 M methanolic HCl and NaOH. The difference absorption spectra of CAN, HYD and their mixture are shown in fig.3a. CAN can be successfully estimated by measuring its [DELTA]A value at 292 nm which corresponds to a zero-crossing for HYD whereas HYD can be determined by measuring its [DELTA]A value at 338 nm which corresponds to a zero-crossing for CAN. Figures 3b and 3c. show the first and second derivative difference spectra of both drugs and their mixture (the influence of [DELTA][lambda] on the first and second derivative of the difference spectra was tested and [DELTA][lambda] = 6 nm was considered suitable for both drugs). In fig. 3b HYD shows a well defined maximum at 299 nm, while CAN has a zero [DELTA][D.sup.1] value at the same wavelength. On the other hand in fig. 3c CAN has a [DELTA][D.sup.2] at 296 nm while HYD exhibits no contribution.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Validation

ICH guidelines [29] for method validation were followed for the developed HPTLC, [DELTA]A and [DELTA]D methods. All validation parameters are shown in tables 1, 2 and 3.

Linearity

The linearity of the proposed methods was evaluated by analyzing series of different concentrations of each of CAN and HYD. According to ICH, at least five concentrations must be used.

Under the experimental conditions described, the graphs obtained by plotting peak area (for HPTLC), [DELTA]A, [DELTA][D.sup.1] and [DELTA][D.sup.2] values versus concentration (in the ranges stated in table 1) show linear relationships. The slopes, intercepts and correlation coefficients obtained by the linear least squares regression treatment of the results are also given. An important statistical parameter for indicating the random error in the estimated values of y is the standard error of the estimate, or the standard deviation about regression, or the standard deviation of the residuals, [S.sub.y/x]. The smaller the standard error of the estimate the closer the points are to the straight line. Standard deviation of intercept ([S.sub.a]) and of slope ([S.sub.b]) are also presented for each compound using the proposed methods of measurements. The high values of the correlation coefficients (r-values > 0.999) with negligible intercepts together with the high F-values indicate the good linearity of the calibration graphs[31, 32]. The linearity was further evaluated by calculation of the percentage relative SD of the slope ([S.sub.b]%). Also, the small degree of scatter of the experimental data point around the line of regressions was confirmed by the small values of the variances around the slopes [S.sub.b.sup.2]. For more confirmation, the Student's t-test was performed to determine whether the experimental intercept (a) of the above-mentioned regression lines was not significantly different from the null hypothesis. The calculated values of t (a/[S.sub.a]) do not exceed the 95% criterion of t = 2.31 for 5 samples. So the intercepts are not significantly different from zero in the proposed methods. Thus, the hypothesis that (a) is of negligible value is confirmed[31, 32].

For equal degrees of freedom, increase in the variance ratio (F-values) means increase in the mean of squares due to regression and decrease in the mean of squares due to residuals. The greater the mean of squares due to regression, the more the steepness of the regression line is. The smaller the mean of squares due to residuals, the less the scatter of the experimental points around the regression line. Consequently, regression lines with high F-values (low significance F) are much better than those with lower ones. Good regression lines show high values for both (r) and (F) values[32].

Limit of Detection and Limit of Quantitation

For HPTLC, limit of detection (LOD) is considered as the concentration which has a signal-to-noise ratio of 3:1. For limit of quantitation (LOQ), the ratio considered was 10:1 with a RSD% value less than 10%[29]. For spectrophotometric methods, LOD and LOQ were calculated using the formulae given by Miller[31] where the limit of detection, LOD = 3 S/b and the limit of quantitation, LOQ = 10 S/b, where S is the standard deviation of replicate blank responses (under the same conditions as for sample analysis) and b is the sensitivity, namely the slope of the calibration graph. Using the proposed methods, LOD and LOQ for each compound were calculated and are presented in table 1.

Accuracy

The accuracy of the proposed methods was evaluated by analyzing five laboratory-prepared mixtures of CAN and HYD at various concentration ratios within the working range of each compound (Table 2). Satisfactory recoveries, small relative errors, with small relative standard deviations (RSD%) were obtained, which indicated the high accuracy of the proposed methods.

Precision

The intra-day and inter-day variation for the determination of CAN and HYD were carried out at three different concentration levels namely; 0.15, 0.50, 0.70 [micro]g x [band.sup.-1] (CAN) and 0.05, 0.25, 0.50 [micro]g.[band.sup.-1] (HYD) for HPTLC. Meanwhile, 20, 60, 100 [micro]g [ml.sup.-1] (CAN) and 20, 50.0, 70.0 [micro]g [ml.sup.-1] (HYD) were used for evaluating the precision of spectrophotometric methods. Method repeatability was obtained from RSD% values obtained by repeating the assay five times on the same day for intra-day precision (Table 3). Intermediate precision was assessed by the assay of the sample sets on three different days (inter-day precision). The RSD% values depicted in Table 3 shows that proposed HPTLC and spectrophotometric methods provides acceptable intra-day and inter-day variation of CAN and HYD.

Robustness

Robustness of the proposed methods was evaluated by analyzing CAN and HYD at the same concentration levels mentioned in 3.3.4 for precision. For HPTLC method, the parameters studied were mobile phase composition, mobile phase volume, duration of saturation, time from chromatography to scanning and different plates. However for spectrophotometric methods, the parameters included, molarity of HCl and NaOH, methanol:water ratio used for preparing methanolic HCl and NaOH solutions, different models of spectrophotometers and different lots of solvent. It was found that variation in the above parameters had no significant influence on the determination of CAN and HYD using the proposed methods. The low values of RSD% of peak areas along with nearly unchanged retardation factor ([R.sub.F]) (for HPTLC method) and the low RSD% of [DELTA]A, [DELTA][D.sup.1] and [DELTA][D.sup.2] values (for spectrophotometric methods) obtained after introducing small deliberate changes in the method parameters indicated the robustness of the developed methods (Table 3).

Selectivity

The selectivity was checked by analyzing synthetic mixtures containing different ratios of both drugs, where good percentage recoveries were obtained indicating that they did not interfere with each other (Table 2). In addition, the application of the proposed method for the determination of the two drugs in dosage forms; without interference from the excipients clearly demonstrate the selectivity of the method (Table 4).

Stability in solutions

The stability of CAN and HYD in their solutions during the analytical procedures was studied. Solutions of the two drugs were prepared and stored at room temperature for 0.5, 1.0, 2 hrs. They were then analyzed using the proposed methods since no additional peaks were found in the chromatograms throughout the analysis time using HPTLC, this indicates the stability of both drugs in the sample solution. Using the spectrophotometric methods, no significant changes in [DELTA]A, [DELTA][D.sup.1] and [DELTA][D.sup.2] values were obtained throughout the analysis time, thus the two drugs are stable in solutions for at least 2 hours.

Furthermore, for HPTLC, Spot stability is very important. The time which the sample is left to stand prior to chromatographic development can influence the stability of separated spots and are required to be investigated for validation [33]. Two-dimensional chromatography using the same solvent system was used to find out any decomposition occurring during spotting and development. In case, if decomposition occurs during development, peak(s) of decomposition product(s) shall be obtained for the analyte both in the first and second direction of the run. Since no decomposition was observed during spotting and development using the proposed conditions, this indicated the stability of drugs in solutions.

Analysis of tablets

The proposed methods were applied to the determination of CAN and HYD in commercial tablets. Five replicate determinations were made. Satisfactory results were obtained for both drugs and were in good agreement with the label claims (Table 4). Moreover, to check the validity of the proposed methods, the standard addition method was applied by adding CAN and HYD to the previously analyzed tablets. The recovery of each drug was calculated by comparing the concentration obtained from the spiked mixtures with those of the pure drug. The results of analysis of the commercial tablets and the recovery study (standard addition method) of both drugs (Table 4) suggested that there is no interference from any tablet excipients, which are present in tablets. The results of determination of CAN and HYD in tablets obtained by the proposed methods were compared with those of the reference spectrophotometric method [27]. A statistical comparison of the results was performed with regard to accuracy and precision using Student's t-test and the F-ratio at a 95% confidence level (Table 4). There is no significant difference between the proposed methods and the reference method with regard to accuracy and precision.

Conclusion

The proposed HPTLC, difference and derivative difference spectrophotometric methods provide simple, accurate, reproducible and specified quantitative analysis for simultaneous determination of candesartan cilexetil and hydrochlorothiazide in tablets. The application of difference spectrophotometry is very advantageous in quantitative and qualitative analysis. The main goal of this method is to increase the selectivity of the measurement by eliminating the effect of foreign light absorption, if the spectra of these substances do not change with the use of different pH values. Moreover, application of the derivative technique to difference spectrophotometry adds to the sensitivity of the method. The HPTLC method has some advantages such as a short run time, large sample capacity and minimal volume use of solvent. With these two methods, one can gain the advantages of speed, lower cost, and environmental protection without sacrificing accuracy.

References

[1] G.H. Cocolas, J.N. Delgado, W.A. Remers, 1998, Textbook of Organic Medicinal and Pharmaceutical Chemistry, tenth ed., Lippincott Raven, Philadelphia, New York.

[2] T. Unger, 1999,"Significance of angiotensin type 1 receptor blockade: why are angiotensin II receptor blockers different?," Am. J. Cardiol., 84, pp. 9S-15S.

[3] R. Willenheimer, B. Dahlof, E. Rydberg, L. Erhardt, 1999,"AT1-receptor blockers in hypertension and heart failure: clinical experience and future directions," Eur. Heart J., 20, pp. 997-1008.

[4] D.V.S. Rao, P. Radhakrishnanand, M.V. Suryanarayan, 2007,"A stability-indicating LC method for candesartan cilexetil," Chromatographia, 66, pp. 499-507.

[5] B. Dogan, B. Uslu, S.A. Ozkan, 2004,"Anodic adsorptive stripping voltammetry of the antihypertensive drug candesartan cilexetil at a glassy carbon electrode.," Die Pharmazie, 59, pp. 840-844.

[6] J.Q. Yuan, S.M. Li, H. Wang, J. Yu, W.T. Jing, 2006,"RP-HPLC determination of candesartan in human plasma with fluorometric detection," Yaowu Fenxi Zazhi, 26, pp. 761-763.

[7] L. Gonzalez, J.A. Lopez, R.M. Alonso, R.M. Jimenez, 2002,"Fast screening method for the determination of angiotensin II receptor antagonists in human plasma by high-performance liquid chromatography with fluorimetric detection.," J. Chromatogr., 949, pp. 49-60.

[8] H. Stenhoff, P.O. Lagerstrom, C. Andersen, 1999,"Determination of candesartan cilexetil, candesartan and a metabolite in human plasma and urine by liquid chromatography and fluorimetric detection.," J. Chromatogr. B:Biomed. Appl., 731, pp. 411-417.

[9] J.W. Lee, N.D. Weng, T. Johnson, A. Dzerk, T. Miyabayashi, M. Motohashi, 1995,"Development and validation of column-switching high-performance liquid-chromatographic methods for the determination of a potent AII receptor antagonist, TCV 116, and its metabolites in human serum and urine," J. Chromatogr. B:Biomed. Appl., 670, pp. 287-298.

[10] The British Pharmacopeia (2007), Her Majesty's Stationery Office, London.

[11] The United States Pharmacopeia, Twenty-ninth edition, The National Formulary, Twenty-fourth edition (2007), United States Pharmacopeial Convention, Inc.

[12] O. Abdel-Razak, 2004,"Electrochemical study of hydrochlorothiazide and its determination in urine and tablets " J. Pharm. Biomed. Anal., 34, pp. 433-440.

[13] L.R. Bhat, R.K. Godge, A.T. Vora, M.C. Damle, 2007,"Validated RP-HPLC method for simultaneous determination of telmisartan and hydrochlorothiazide in pharmaceutical formulation," J. Liq. Chrromatogr. Rel. Tech., 30, pp. 3059-3067.

[14] Y.Q. Wei, H.B. Xou, Y.J. R., 2006,"Simultaneous LC determination of chlorogenic acid and hydrochlorthiazide in Zhenju Jiangya tablets," Chromatographia, 64, pp. 709-711.

[15] S. Erturk, S.M. Centin, S. Atmaca, 2003,"Simultaneous determination of moexipril hydrochloride and hydrochlorothiazide in tablets by derivative spectrophotometric and high-performance liquid chromatographic methods," J. Pharm. Biomed. Anal., 33, pp. 505-511.

[16] E. Dinc, O. Ustundag, 2005,"Application of multivariate calibration techniques to HPLC data for quantitative analysis of a binary mixture of hydrochlorathiazide and losartan in tablets," Chromatographia, 61, pp. 237-244.

[17] D. Kowalczuk, H. Hopkala, R.J. Pietras, 2003,"Simultaneous densitometric determination of quinapril and hydrochlorothiazide in the combination tablets," J. Planar Chromatogr. Mod. TLC, 16, pp. 196-200.

[18] S.A. Shah, I.S. Rathod, B.N. Suhagia, S.S. Savale, J.B. Patel, 2001,"Simultaneous determination of losartan and hydrochlorothiazide in combined dosage forms by first-derivative spectroscopy and high-performance thin-layer chromatography," J.AOACInt., 84, pp. 1715-1723.

[19] D. Kowalczuk, H. Hopkala, 2004,"Application of derivative spectrophotometry for simultaneous determination of quinapril and hydrochlorothiazide in the combination tablets," J.AOAC Int., 87, pp. 847-851.

[20] E. Dinc, B. Uslu, S.A. Ozkan, 2004,"Spectral Resolution Of A Binary Mixture Containing Valsartan And Hydrochlorothiazide In Tablets By Ratio Spectra Derivative And Invers Least Square Techniques," Anal. Lett., 37, pp. 679-693.

[21] N. Erk, 2003,"Three new spectrophotometric methods applied to the simultaneous determination of hydrochlorothiazide and irbesartan," Die Pharmazie, 58, pp. 543-548.

[22] T.M. Huang, Z. He, B. Yang, L.P. Shao, X.W. Zheng, G.L. Duan, 2006,"Simultaneous determination of captopril and hydrochlorothiazide in human plasma by reverse-phase HPLC from linear gradient elution.," J. Pharm. Biomed. Anal., 41, pp. 644-648.

[23] A. Vonaparti, M. Kazanis, I. Panderi, 2006,"Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the determination of benazepril, benazeprilat and hydrochlorothiazide in human plasma," J. Mass Spect., 41, pp. 593-605.

[24] A.A. El-Emam, S.H. Hansen, M.A. Moustafa, S.M. El-Ashry, D.T. El Sherbiny, 2004,"Determination of lisinopril in dosage forms and spiked human plasma through derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) followed by spectrophotometry or HPLC with fluorimetric detection," J. Pharm. Biomed. Anal., 34, pp. 35-44.

[25] N. Erk, 2003,"Simultaneous Analysis of Candesartan Cilexetil and Hydrochlorothiazide in Human Plasma and Dosage Forms Using HPLC with a Photodiode Array Detector " J. Liq. Chrromatogr. Rel. Tech., 26, pp. 2581-2591.

[26] J. Lei, X.S. Zhang, Z. Zhuo, K. Zhu, P. Sun, Q. Fan, 2007,"HPLC-UV simultaneous determination of candesartan cilexetil and hydrochlorothiazide in compound candesartan cilexetil tablets," Yaowu Fenxi Zazhi, 27, pp. 566-568.

[27] N. Erk, 2003,"Application of first derivative UV-spectrophotometry and ratio derivative spectrophotometry for the simultaneous determination of candesartan cilexetil and hydrochlorothiazide.," Die Pharmazie, 58, pp. 796-800.

[28] S. Hillaert, W. Van Den Bossche, 2003,"Simultaneous determination of Hydrochlorothiazide and several angiotensin-II-receptor antagonists by capillary electrophoresis," J. Pharm. Biomed. Anal., 31, pp. 329-339.

[29] I. Q2A, Q2A (R1), Validation of Analytical Procedures: Text and Methodology, International Conference on Harmonization, Geneva, (November 2005), (http://ww.ich.org/LOB/media/MEDIA417.pdf),

[30] A.C. Moffat, M.D. Osselton, B. Widdop, 2004, Clarke's Analysis of Drugs and Poisons, thirrd edition, London.

[31] J.N. Miller, J.C. Miller, 2000, Statistics and Chemometrics for Analytical Chemistry, 4th ed., Prentice Hall, Harlow, England.

[32] P. Armitage, G. Berry, 1994, Statistical Methods in Medical Research, 3 rd ed., Blackwell Scientific Publications, Oxford, England.

[33] P.D. Sethi, 1996, High Performance Thin Layer Chromatography: Quantitative Analysis of Pharmaceutical Formulations, CBS Publishers and Distributors, New Delhi, India.

Rasha M. Youssef (a) *, Hadir M. Mahera, Ekram M. Hassan (a,b), Eman I. El-Kimary (a) and Magda A. Barary (a)

(a) Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, University of Alexandria, El-Messalah, Alexandria 21521, Egypt

(b) Faculty of Pharmacy, Department of Pharmaceutical Analytical Chemistry, Beirut Arab University

E-mail: rmmy1973@yahoo.com

Table 1: Regression and statistical parameters for the determination of CAN and HYD using the proposed methods. Parameters HPTLC CAN HYD Linearity range ([micro]g 0.05-0.70 * 0.05-0.50 * [ml.sup.-1]) LOQ ([micro]g [ml.sup.-1]) 0.045 * 0.035 * LOD ([micro]g [ml.sup.-1]) 0.014 * 0.010 * Intercept 45.96 103.38 Slope 9.907 25.89 Correlation coefficient 0.9999 0.9997 [S.sub.a] 22.68 118.79 [S.sub.b] 0.0523 0.399 [S.sub.y/x] 28.49 129.21 a/[S.sub.a]** 2.03 0.87 [S.sub.b.sup.2] 2.74 x [10.sup.-3] 0.1592 [S.sub.b]% 0.53 1.54 F 35902.22 4216.02 Significance F 2.75 x [10.sup.-5] 2.37 x [10.sup.-4] Parameters Difference spectrophotometry CAN A[[DELTA].sub.292] [DELTA] [D.sup.2.sub.292] Linearity range ([micro]g 20-100 10-100 [ml.sup.-1]) LOQ ([micro]g [ml.sup.-1]) 7.15 4.87 LOD ([micro]g [ml.sup.-1]) 2.15 1.46 Intercept 0.0014 -4.48 x [10.sup.-3] Slope 2.89 x [10.sup.-3] 0.717 Correlation coefficient 0.9995 0.9997 [S.sub.a] 0.00345 3.77 x [10.sup.-3] [S.sub.b] 5.20 x [10.sup.-5] 0.0105 [S.sub.y/x] 3.29 x [10.sup.-3] 5.48 x [10.sup.-3] a/[S.sub.a]** 0.41 1.19 [S.sub.b.sup.2] 2.70 x [10.sup.-9] 1.10 x [10.sup.-4] [S.sub.b]% 1.8 1.46 F 3093.37 4581.86 Significance F 1.28 x [10.sup.-5] 7.11 x [10.sup.-6] Parameters Difference spectrophotometry HYD [DELTA][A.sub.338] [DELTA] [D.sup.1.sub.299] Linearity range ([micro]g 20-100 May-1970 [ml.sup.-1]) LOQ ([micro]g [ml.sup.-1]) 6.22 2.55 LOD ([micro]g [ml.sup.-1]) 1.87 0.77 Intercept -2.40 x [10.sup.-3] -6.73 x [10.sup.-3] Slope 4.03 x [10.sup.-3] 1.222 Correlation coefficient 0.9993 0.9998 [S.sub.a] 5.60 x [10.sup.-3] 5.69 x [10.sup.-3] [S.sub.b] 8.45 x [10.sup.-5] 0.0139 [S.sub.y/x] 5.34 x [10.sup.-3] 7.57 x [10.sup.-3] a/[S.sub.a]** 0.43 1.18 [S.sub.b.sup.2] 7.14 x [10.sup.-9] 1.93 x [10.sup.-4] [S.sub.b]% 2.1 1.14 F 2276.76 7755.45 Significance F 2.03 x [10.sup.-5] 3.23 x [10.sup.-6] [S.sub.a] is standard deviation of intercept, [S.sub.b] is standard deviation of slope, and [S.sub.y/x] is standard deviation of residuals * Concentration in [micro]g x [band.sup.-1] ** Theoretical value of t (a/[S.sub.a]) = 2.31 at the 95% confidence level Table 2: Analysis of CAN and HYD in synthetic mixtures using the proposed methods. a) HPTLC method CAN:HYD Mean % recovery [+ or -] SD (a) [micro]g x [band.sup.-1] CAN HYD 0.32:0.25 100.20 [+ or -] 0.886 99.69 [+ or -] 0.748 0.5:0.5 100.76 [+ or -] 1.098 100.34 [+ or -] 1.196 0.4:0.1 99.84 [+ or -] 0.400 100.44 [+ or -] 1.140 0.2:0.5 100.93 [+ or -] 0.586 101.01 [+ or -] 0.824 0.5:0.3 99.25 [+ or -] 0.965 99.61 [+ or -] 0.722 CAN:HYD RSD % (b) Er (%) (c) [micro]g. [band.sup.-1] CAN HYD CAN HYD 0.32:0.25 0.884 0.750 0.20 -0.31 0.5:0.5 1.090 1.192 0.76 0.34 0.4:0.1 0.401 1.135 -0.16 0.44 0.2:0.5 0.581 0.816 0.93 1.01 0.5:0.3 0.972 0.725 -0.75 -0.39 (b) Difference spectrophotometry CAN:HYD Mean % recovery [+ or -] SD (a) [micro]g [ml.sup.-1] CAN HYD [DELTA] [DELTA] [DELTA] [A.sub.292] [D.sup.2.sub.296] [A.sub.338] 48:37.5 99.27 100.83 99.78 [+ or -] 0.635 [+ or -] 1.440 [+ or -] 0.372 60:60 100.55 99.91 100.72 [+ or -] 0.721 [+ or -] 0.894 [+ or -] 0.444 5:15 99.97 101.01 100.12 [+ or -] 0.616 [+ or -] 0.686 [+ or -] 0.496 10:5 99.34 100.21 99.13 [+ or -] 0.582 [+ or -] 0.462 [+ or -] 0.772 30:20 99.18 99.18 99.36 [+ or -] 0.626 [+ or -] 0.972 [+ or -] 0.528 CAN:HYD Mean % recovery RSD % (b) [micro]g [+ or -] SD (a) [ml.sup.-1] HYD CAN [DELTA] [DELTA] [DELTA] [D.sup.1.sub.299] [A.sub.292] [D.sup.2.sub.296] 48:37.5 99.21 0.640 0.884 [+ or -]1.374 60:60 99.27 0.717 1.090 [+ or -] 0.780 5:15 99.34 0.616 0.401 [+ or -] 0.778 10:5 99.30 0.586 0.581 [+ or -] 0.254 30:20 100.93 0.631 0.972 [+ or -] 1.125 CAN:HYD RSD % (b) Er(%) (c) [micro]g [ml.sup.-1] HYD CAN [DELTA] [DELTA] [DELTA] [A.sub.338] [D.sup.1.sub.299] [A.sub.292] 48:37.5 0.373 1.428 -0.73 60:60 0.441 0.895 0.550 5:15 0.495 0.679 -0.03 10:5 0.779 0.461 -0.66 30:20 0.531 0.980 -0.82 CAN:HYD Er(%) (c) [micro]g [ml.sup.-1] CAN HYD [DELTA] [DELTA] [DELTA] [D.sup.2.sub.296] [A.sub.338] [D.sup.1.sub.299] 48:37.5 0.83 -0.22 -0.79 60:60 -0.09 0.72 -0.73 5:15 1.04 0.12 -0.66 10:5 0.21 -0.87 -0.70 30:20 -0.82 -0.64 0.93 (a) Mean [+ or -] standard deviation of three determinations. (b) Percentage relative standard deviation. (c) Percentage relative error. Table 3: Precision and robustness of the proposed methods. Parameters CAN (I) HPTLC (a) SD of RSD% [R.sub.F] [+ or -] SD peak areas * Precision Intra-day 219 1.46 -- Inter-day 285 1.90 -- * Robustness 1) Mobile phase composition [chloroform : methanol (8.2: 1.8, 8.0: 2.0 and 7.8: 2.2 219 1.75 0.73 [+ or -] 0.015 v/v)] 2) Mobile phase volume [15, 187 1.50 0.74 [+ or -] 0.010 20 and 25 mL] 3) duration of saturation 79 0.63 0.75 [+ or -] 0.006 [20, 30 and 40 min] 4) Time from chromatography 46 0 37 0 74 [+ or -] 0 005 to scan. [5, 10 and 20 min] 46 0.37 0.74 [+ or -] 0.005 5) Plates from different manufactures. [E-Merk and 236 1.89 0.73 [+ or -] 0.014 Fluka] Parameters HYD (I) HPTLC (a) SD of RSD% [R.sub.F] [+ or -] SD peak areas * Precision Intra-day 231 1.02 -- Inter-day 417 1.85 -- * Robustness 1) Mobile phase composition [chloroform : methanol (8.2: 1.8, 8.0: 2.0 and 7.8: 2.2 392 1.87 0.46 [+ or -] 0.009 v/v)] 2) Mobile phase volume [15, 282 1.35 0.44 [+ or -] 0.008 20 and 25 mL] 3) duration of saturation 234 1.12 0.45 [+ or -] 0.006 [20, 30 and 40 min] 4) Time from chromatography 1 59 0 76 0 46 [+ or -] 0 003 to scan. [5, 10 and 20 min] 159 0./6 0.46 [+ or -] 0.003 5) Plates from different manufactures. [E-Merk and 406 1.94 0.45 [+ or -] 0.009 Fluka] (II) Difference CAN spectrophotometry (b) * Precision SD of RSD% [DELTA] [A.sub.292] Intra-day 0.011 1.47 Inter-day 0.014 1.87 * Robustness 1) Molarity of HCl and NaOH 0.006 1.14 (0.05, 0.06, 0.07 M) 2) Methanol:water ratio in preparation of 0.06M methanolic NaOH or 0.007 1.33 methanolic HCl (3:7, 4:6, 5:5 v/v) 3) Spectrophotometer of 0.010 1.89 different models * 4) Methanol of different lots. 0.004 0.76 (II) Difference CAN spectrophotometry (b) * Precision SD of [DELTA] RSD% [D.sup.2.sub.296] Intra-day 0.014 0.96 Inter-day 0.025 1.72 * Robustness 1) Molarity of HCl and NaOH 0.019 1.57 (0.05, 0.06, 0.07 M) 2) Methanol:water ratio in preparation of 0.06M methanolic NaOH or 0.008 0.69 methanolic HCl (3:7, 4:6, 5:5 v/v) 3) Spectrophotometer of 0.021 1.73 different models * 4) Methanol of different lots. 0.011 0.91 (II) Difference HYD spectrophotometry (b) * Precision SD of RSD% [DELTA] [A.sub.338] Intra-day 0.016 1.57 Inter-day 0.021 1.96 * Robustness 1) Molarity of HCl and NaOH 0.012 1.67 (0.05, 0.06, 0.07 M) 2) Methanol:water ratio in preparation of 0.06M methanolic NaOH or 0.011 1.53 methanolic HCl (3:7, 4:6, 5:5 v/v) 3) Spectrophotometer of 0.014 1.95 different models * 4) Methanol of different lots. 0.008 1.12 (II) Difference HYD spectrophotometry (b) * Precision SD of [DELTA] RSD% [A.sup.1.sub.292] Intra-day 0.008 0.53 Inter-day 0.020 1.40 * Robustness 1) Molarity of HCl and NaOH 0.018 1.42 (0.05, 0.06, 0.07 M) 2) Methanol:water ratio in preparation of 0.06M methanolic NaOH or 0.013 1.03 methanolic HCl (3:7, 4:6, 5:5 v/v) 3) Spectrophotometer of 0.023 1.82 different models * 4) Methanol of different lots. 0.009 0.71 (a) Average of three concentrations 0.05, 0.50, 0.70 [micro]g x [band.sup.-1] and 0.05, 0.25, 0.50 [micro]g [band.sup.-1] for CAN and HYD, respectively. (b) Average of three concentrations 20, 60, 100 [micro]g [ml.sup.-1] and 20, 50.0, 70.0 [micro]g [ml.sup.-1] for CAN and HYD, respectively. * A ThermoSpectronic UV-vis spectrophotometer connected to Harvest computer system and Perkin-Elmer Lambda EZ201 UV-visible spectrophotometer Table 4: Assay of CAN and HYD in tablets using the proposed HPTLC and difference spectrophotometric methods. Atacand CAN -plus[R] (a) HPTLC [DELTA] [DELTA] Reference [A.sub.292] [D.sup.2.sub.296] method[27] Mean % 98.80 99.01 100.36 99.90 recovery [+ or -] [+ or -] [+ or -] 1.181 [+ or -] [+ or -] 0.751 0.901 1.560 SD (b) RSD (%) 0.487 0.910 1.177 1.562 Er (%) -1.20 -0.99 0.36 -0.10 t (c) 0.67 1.11 0.53 -- F (c) 4.31 2.99 1.71 -- Recovery study (d) Mean % 99.61 99.15 100.61 -- recovery [+ or -] [+ or -] [+ or -] 0.834 [+ or -] 0.771 0.614 SD Atacand HYD -plus[R] (a) HPTLC [DELTA] [DELTA] Reference [D.sup.1.sub.299] [A.sub.338] method[27] Mean % 99.62 99.91 [+ or -] 100.05 99.32 recovery [+ or -] 0.515 [+ or -] [+ or -] [+ or -] 0.804 0.915 0.484 SD (b) RSD (%) 0.807 0.515 0.915 0.487 Er (%) -0.38 -0.09 0.15 -0.68 t (c) 0.71 1.87 1.68 -- F (c) 2.76 1.13 3.57 -- Recovery study (d) Mean % 100.71 99.45 100.59 -- recovery [+ or -] [+ or -] 0.372 [+ or -] [+ or -] 0.523 1.116 SD (a) labeled to contain labeled to contain 12.5 mg HYD and 16 mg CAN/tablet (Batch No. HA4474) (b) Mean [+ or -] standard deviation of five determinations. (c) Theoretical values of t and F are 2.31 and 6.39, respectively, at 95% confidence limit. (d) For standard addition of 50% of the nominal content

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Title Annotation: | thin layer chromatography |
---|---|

Author: | Youssef, Rasha M.; Maher, Hadir M.; Hassan, Ekram M.; El-Kimary, Eman I.; Barary, Magda A. |

Publication: | International Journal of Applied Chemistry |

Article Type: | Report |

Geographic Code: | 7EGYP |

Date: | May 1, 2010 |

Words: | 5985 |

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