Hiqh-Perfonnance Thin-Laver C hrornatoaraphv for the Determination of Certain Antihypertensive Mixtures

A new simple, precise, accurate and selective high-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous deter-mination of five mixtures: atenolol and chlorthalidone (Mix. l), enalapril maleate and hydrochlorothiazide (Mix. ll), amiloride hydrochloride and hydrochlorothiazide (Mix. lll), atenolol, chlorthalidone and amiloride hydrochloride (Mix. lV) and atenolol, hydrochlorottriazide and amiloride hydrochloride (Mix. V) in bulk powders and in pharmaceutical dosage forms. The methods consist of dissolving the drugs in methanol and spotting these solutions on a thin layer Merck HPTLC plates (0.25 mm thickness) pre-coated wrth 60 GF254 silica gel on aluminum sheet as the stationary phase, using dioxane:acetonrtrile: 1-propanol:hexane (30:18:23:1; v/v/v/v) , ethylacetate:chlo-roform:methanol:acetic acid (11:8:7.5:1.5; v/v/v/v). ethylacetate:chloroform:1-propanol:25% ammonia solution (12:9:1:0.2; v/v/v/v), di0xan:acetonitrile:1-propanol:tetrahydrofuran (20:13:4:15; v/v/v/v) and dioxane:ethylacetate:acetonitrile:1-propanol (10:7:5.5:3 v/v/v/v) as the mobile phases for mixtures l, ll, lll, lV and V, respectively. Detection was carried out densitometrically using UV detector at 283, 266, 257, 226 and 362 for atenolol, chlorthalidone, enalapril maleate, hydrochlorothiazide and amiloride hydrochloride, respectively. Calibration curves were linear in the range 1–100 µg/ml−1 with correlation coefficients not less than 0.9996. The percentage recoveries ranged from 98.3 ± 1.42 to 100.8 ± 0.79.


NH2
Atenolol is a beta-adrenergic receptor blocking agent, indicated in the management of hypertension [9].Voltametric [lo], spectrophotometric [ I 1-1 31 and chromatographic [14-161 techniques have been used for the assay of atenolol.
Chlorthalidone is a thiazide derivative.It has a very long duration of action, therefore it is often used to treat hypertension [ A T ] .Fluorometric [18], and chromatographic [ I 9-24] techniques have been employed for determination of chlorthalidone.In the present paper, HPTLC has been recommended for the assay of mixtures of the above-mentioned drugs in laboratory prepared mixtures and in pharmaceutical dosage forms.
H. S a l e m :

P)rsnnsceutical prsparPtions
The commercial dosage forms subpcted to analysis were Tenedone 100@ tablets.labeled to contain 50 mg atenolol, 25 mg hydrochlorothiazide and 2.5 mg amiloride hydrochloride.

I . Constnrction of cdibmtbn curves
Aliquot portions from each stock solution were transferred into 10 ml calibrated flasks.The flasks were made up to volume with methanol to form working solutions, which  Record the area under the peaks and calculate the concentration of drugs from the regression equation or referring to calibration curves.

Assay oftsMeb and c q x w k
The contents of twenty tablets and capsules of the drugs were thoroughly ground.A quantrty equivalent to the labeled active constituents was accurately weighed into a 100 ml volumetric flasks, completed to volume with the appropriate solvent and the procedure was completed as under 3.1.and 3.2.

Resufts and discussion
In the present paper.spectrodensitmetry was examined for the assay of mixtures of the above-mentioned drugs.
The proposed method was used for separating atenolol from chlorthalidone (Mix.I).
enalapril maleate from hydrochlorothiazide (Mix.I I), amiloride hydrochloride from hydrochlorothiazide (Mix.Ill), atenolol from chlorthalidone and from amiloride hydrochloride (Mix.IV) and atenolol from hydrochlorothiazide and from amiloride hydrochloride (Mix.V).The m e h d depends on the difference of the Rfvalues of the analyzed drugs (Table 1).Various common excipients and coloring matter did not interfere in the separation process.

Linear mgmdcm ecptbns
A calibration graph (peak area ratio versus concentration) for each component was For mixture V: Y = 0.575 + 6.99 X (r = 0.9996) for atenolol Y = 0.799 + 10.21 X (r = 0.9997) for hydrochlorothiazide Y = 0.600 + 3.98 X (r = 0.9999) for amiloride hydrochloride Where X is the concentration of related drug (pglspot), r is the correlation coefficient and Y is the area under the peak.

Statistical analysis
Statistical analysis of the results obtained by the proposed methods are compared wrth results of the reported and official methods [37 and 401 (Tables 2-6) at a 95% confidence level, the calculated t and F values do not exceed the tabulated ones, revealing equal precision and accuracy.

Validity
The validity of the proposed method was also checked by applying the standard addition technique.(Tables 7-1 1 ).

Conclusion
From the above study, it is concluded that the proposed method is highly sensitive.
Because a variable wavelength UV detector can be used.each substance can be determined of very low concentration of each substance.Furthermore, the method is rapid and less expensive than HPLC method.
indicated for the treatment of hypertension.It is effective alone or in combination wtth other antihypertensive agents [25].Several methods have been reported for determination of enalapril maleate.Recent methods include, spectrophotornetric [26,27J and chromatographic [28-301 techniques.Hydrochlorothiazide is one of wellknown thiazide diuretics, it counteracts the sodium and water retention observed with other agents used in treatment of hypertension High-Performance Thin-Layer Chromatography for the Determination of Certain . . .159 [I].Several methods have been reported for determination of hydrochlorothiazide include, spectrophotometric [31-331 and chromatographic [34-361 techniques.Only two techniques were reported for the simultaneous determination of the above mixtures, the first one is a HPLC technique used for separation of atenolol from chlorthalidone [37.381, enalapril rnaleate from hydrochlorothiazide [37, 391 and arniloride hydrochloride from hydrochlorothiazide [37l.The second one is a derivative spectrophotometric technique used for analysis for all the studied mixtures[40].
contain 0.5 mg rnl-' of the cited drugs.1-5.5-25.20-100.4-20 and 10-50 pg for amiloride hydrochloride, chlorthalidone, atenolol, enalapril maleate and hydrochlorothiazide, respectively, are accurately transferred to thin layer chromatographic plates (20 x 20 crn) using 25 pl Hamilton syringe.Spots are spaced 2 cm apart from each other and 2 cm from the bottom edge of the plate.The plates are developed in chromatographic tank previously H. S a l e m : saturated at least two hours with the developing mobile phases (I.11.Ill.IV and V), by ascending chromatography through a distance of 6 cm at room temperature.The spots were air dried and measured at suitable wavelengths 283, 266, 257,226 and 362 for atenolol, chlorthalidone, enalapril maleate, hydrochlorothiazide and amiloride hydrochloride, respectively.The drugs under reflection photo mode and zigzag scan mode conditions are scanned.The areas under the peaks are recorded.The calibration curves are constructed by plotting area under the peak versus the corresponding concentrations.

Table 2 HFTLC analysis of a6ecrdd and chlorth;rlidorre (Mix. I), in a laborabory prepared mixture and in phamaceuthl closage forms.
High-Performance Thin-Layer Chromatography for the Determination of Certain . . .16hree and six determinations were used for the reported and the HPTLC methods.respect~vely The tabulated values o f t and F at 95% confidence limit are t=2.23 and F=5.79.

HPTLC analysis of milorids hydrochloride and hydrochlorothiazide (Mix. Ill), in a laboratory prepared mixture and in pharmaceutical dosage fonns.
Three and SIX determinations were used for the reported and the HPTLC methods.respectively.The tabulated values o f t and F at 95% confidence limit are t=2.23 and F=5 79

Table 10 Application of the standard addition technique to the analysis of fekloB tablets.
High-Performance Thin-Layer Chromatography for the Determination of Certain . . .169

Table 1 1 Application of the standard addition technique to the analysis of Atenoretic
@ capsu)es.