Analytical Spectrophotometric Study for Determining Hydrocholrothiazide individually or with Combination with Triametrene in some Pharmaceutical Formulations

 

Saad Antakli, Leon Nejem, Ahmad Kullah

Department of Chemistry, Faculty of Science, University of Aleppo, Syria.

 

ABSTRACT:

Spectrophotometric method was developed and applied for simultaneous determination of Hydrochlorothiazide (HYD) individually or with combination with Triamterene (TRM) in some pharmaceutical formulations. This new method was applied to determine (HYD) and (TRM) in several Syrian trademark drugs such as: (Biazid 50 mg (TRM) and 25 mg (HYD)/tab, Hydrochlorothiazide Alfares 25 mg/tab, Hydrochlorothiazide Alfares 12.5 mg/tab, DIUREX 25 mg/tab). Derivative spectrophotometric (DS) method was applied for the determination of (HYD) and (TRM), respectively. (HYD) was determined by first derivative spectrophotometry at 280.5 nm (1D 280.5), and (TRM) was determined by first derivative spectrophotometry at 379 nm (1D 379). Linearity ranges were 0.64 – 28.8 𝜇g/mL for (HYD) and 0.8 – 19.2 𝜇g/mL for (TRM), regression analysis showed a good correlation coefficients R² = 0.9997 and R² = 0.9999 for (HYD) and (TRM), respectively. The limit of detection (LOD) and limit of quantification (LOQ) were to be 0.143 𝜇g/mL and 0.433 𝜇g/mL for (HYD), 0.128 𝜇g/mL and 0.390 𝜇g/mL for (TRM), respectively. 0.134 in table 1 The proposed derivative method was successfully applied to analysis individual or mixture of (HYD) and (TRM) in Syrian trademark drugs. All studied samples showed that the drug levels were conformed to united state pharmacopeia (USP) legislation.

 

 

 

INTRODUCTION:

Hydrochlorothiazide (HYD) is a diuretic medication often used to treat high blood pressure and swelling due to fluid buildup. Other uses include diabetes insipidus, renal tubular acidosis, and to decrease the risk of kidney stones in those with high calcium level in the urine, and to decrease high blood pressure.

 

Triamterene (TRM) is commonly prepared in combination with (HYD) for treatment of hypertension (high blood pressure) and edema (water retention). This combination is in a class of medications called diuretics or 'water pills', and causes the kidneys to get rid of the body's unneeded water and sodium through the urine.

 

Triamterene (TRM) (2, 4, 7-tri-amino-6-phenylpteridine) is used commonly in combination with Hy­drochlorothiazide (HYD) (6chloro-3, 4-dihydro-2H-1, 2, 4benzothiadiazine-7-sulphonamide1, 1-dioxide). The determination of these drugs is a frequent analyti­cal problem in quality control of the pharmaceuti­cal industries. The two drugs were studied in this work showed a strong overlap between their absorption spectra. Hence, their simultaneous determination is hard when conventional spectrophotometric techniques are used. Normally, the method used to resolve a complex mixture of these drugs is mainly¹, so we used the spectral derivation method to determine the two compounds.

 

Thus, various methods have been proposed to determine the amount of (HYD) individually and the two compounds in some pharmaceutical formulations, such as thin-layer chromatography (TLC)², reverse phase high performance liquid chromatographic method^3,4 (RP-HPLC) and amperometric detection⁵. Spectrophotometric and HPLC⁶, derivative Spectrophotometricmethods^7-12, Spectrophotometric^13,14. Are successfully applied to determine the two compounds.

 

The aim of this work is to develop a simple and accurate spectrophotometric method for simultaneous determination of (HYD) Fig.1, (TRM) Fig. 2 and their mixtures in some pharmaceutical formulations without prior treatment by UV/Vis spectrophotometry and derivative spectrophotometry (DS).

 

 

Fig. 1. Structural formula of (HYD)

Fig. 2. Structural formula of (TRM)

 

MATERIALS AND METHODS:

Apparatus:

All spectral measurements were carried out using a T80+ UV/VIS spectrophotometer PG instrument Ltd (UK) connected to computer; quartz cells 1 cm. Ultrasonic bath (Daihan) China, and stirrer Velp Scientifica (Europe).

 

Chemical regents:

Double distilled water, Hydrochloric acid from SURCHEM PRODUCTS LTD (ENGLAND), Methanol from LOBAL Chemie (INDIA), Hydrochlorothiazide (20140302) purity 99.38 %, was obtained from China and Triamterene (863-MC) purity 100.57 % was obtained from (MOEHS) Spain.

 

Stock standard preparation:

Stock solutions (400 𝜇g/mL) of (HYD) and (TRM) were prepared by dissolving appropriate weights of pure material in methanol. Taking the purity of the material on consideration, the weight became (402.49 µg /mL) and (400 µg /mL), for (HYD) and (TRM), respectively. The working standard solutions of each pharmaceutically pure samples were prepared by appropriate dilutions of stock solutions with HCl (0.1N) to give concentrations between 0.16-32 𝜇g/mL of (HYD) and 0.16-22.4 𝜇g/mL of (TRM).

 

Calibration Curve:

To construct the calibration curve, five standard solutions for each concentration were prepared and the absorbance was measured of each solution five times.

 

 

Sample preparations:

Four Syrian products were studied:

·         Ten BIAZID tablets were weighed and finely powdered and an accurate weight equivalent to one tablet 50 mg (TRM) and 25 mg (HYD) was accurately weighed dissolved in a mixture of 50 mL methanol and 50 mL HCl (0.1N). The sample solution was filtered through a filter papers 3 (Whatman, England). Then 0.32 mL was taken to 25 mL volumetric flask and adjusted to volume with HCl (0.1N), equivalent to 3.2 𝜇g/mL for (HYD) and 6.4 𝜇g/mL for (TRM).

·         Ten (HYD) Alfares 12.5 mg tablets were weighed and finely powdered and an accurate weight equivalent to one tablet 12.5 mg (HYD) was accurately weighed, dissolved in a mixture of 25 mL methanol and 25 mL HCl (0.1N). The sample solution was filtered through a filter papers 3 (Whatman, England). Then 0.60 mL was taken to 25 mL volumetric flask and adjusted to volume with HCl (0.1N), equivalent to 6 𝜇g/mL for (HYD).

·         Ten (HYD) Alfares 25 mg tablets were weighed and finely powdered, and an accurate weight equivalent to one tablet 25 mg (HYD) was accurately weighed, dissolved in a mixture of 25mL methanol and 25 mL HCl (0.1N). The sample solution was filtered through a filter papers 3 (Whatman, England). Then 0.40 mL was taken to 25 mL volumetric flask and adjusted to volume with HCl (0.1N), equivalent to 8 𝜇g/mL for (HYD).

·         Ten DIUREX 25mg tablets were weighed and finely powdered, and an accurate weight equivalent to one tablet 25 mg (HYD) was accurately weighed, dissolved in a mixture of 25 mL methanol and 25 mL HCl (0.1N). The sample solution was filtered through a filter papers 3 (Whatman, England). Then 0.40 mL was taken to 25 mL volumetric flask and adjusted to volume with HCl (0.1N), equivalent to 8 𝜇g/mL for (HYD).

 

RESULTS AND DISCUSSION:

Absorption spectra of the standard pharmaceutically pure samples 6.4 𝜇g/mL (HYD) and 12.8 𝜇g/mL (TRM) solutions was recorded within a wavelength range of 250–400 nm against the blank (all the addition constituents without (TRM) and (HYD)), Fig. 3. As can be seen, (HYD) cannot be determined by direct measurement of absorbance at 271.5 nm, and (TRM) cannot too, because of the overlapped spectra.

 

 

 

 

 

 

 

 

 

 

 

Fig. 3. Zero-order spectra of: a- (HYD), b- (TRM), c- Mixture of (HYD) and (TRM)

 

 

Fig. 4. First derivative spectra of: a- (HYD), b- (TRM).

 

On the other hand, derivative spectrophotometry showed more resolution. Where it made the determination of (HYD) and (TRA) mixture possible without pretreatment.

 

The first derivative spectrum at zero-crossing point was used to determine (HYD) in the presence of (TRA) at 280.5 nm (Fig. 4, a).

 

The first derivative spectrum at zero-crossing point was used to determine (TRA) in the presence of (HYD) at 379 nm (Fig. 4, b).

 

METHOD VALIDATION:

The validity of the proposed method was assessed by accuracy (reported as recovery percentage), precision (reported as RSD %), linearity (evaluated by regression equation), limit of detection (LOD) and limit of quantification (LOQ).

 

Linearity:

The concentration linearity of (HYD) was in the range 0.64-28.8 𝜇g/mL at 280.5 nm by ¹D_280.5 Figs 5, 6 and the concentration linearity of (TRM) was in the range 0.8-19.2 µg/mL at 379 nm by ¹D₃₇₉ Figs. 7, 8.

 

 

Fig. 5: First derivative spectra of (HYD):

C₁: 0.64 𝜇g/mL, C₂: 0.8 𝜇g/mL, C₃: 1.6 𝜇g/mL,

C₄: 3.2 𝜇g/mL, C₅: 6.4 𝜇g/mL, C₆: 9.6 𝜇g/mL,

C₇: 12.8 𝜇g/mL, C₈: 16 𝜇g/mL, C₉: 19.2 𝜇g/mL,

C₁₀: 22.4 𝜇g/mL, C₁₁: 25.6 𝜇g/mL

C₁₂: 28.8 𝜇g/mL

 

Fig. 6: Calibration curve for (HYD)

n=5 for each concentration.

 

 

Fig.7: First derivative spectra of (TRM):

C1: 0.8 𝜇g/mL, C2: 1.6 𝜇g/mL, C3: 3.2 𝜇g/mL, C4: 6.4 𝜇g/mL, C5: 9.6 𝜇g/mL, C6: 12.8 𝜇g/mL C7: 16 𝜇g/mL, C8: 19.2 𝜇g/mL.

 

Limit of Detection (LOD) and Limit of Quantification (LOQ):

LOD and LOQ were calculated in Table 1 using the following equations:

 

LOQ = 10 × SD

                         M

 

LOD = 3.3 × SD

                       m

Where SD is the standard deviation of y-intercepts (a) of regression lines and (b) is the slope of the equitation of calibration curve, y = a + b x.

 

 

Fig. 8: Calibration curve for (TRM).

n=5 for each concentration.

 

Table 1: Statistical data for calibration graphs.

Method	Analyte	Selected wavelength (nm)	Linearity rang 𝜇g/mL	Correlation coef. (R2)	LOD 𝜇g/mL	LOQ 𝜇g/mL
DS	HYD	1D 280.5	0.64 - 28.8	0.9997	0.143	0.433
DS	TRM	1D 379.0	0.80 - 19.2	0.9999	0.128	0.390

 

Accuracy and precision:

To determine the precision and accuracy of the proposed methods, five replicate determinations were carried out on three different concentrations of standards (HYD) and (TRM). The validation results are shown in Table 2.

 

 

 

Table 2: Method validation for the simultaneous determination of Hydrochlorothiazide and Triamterene by the proposed methods.

Method	Pharmaceutically pure samples	Theoretical concentration (μg/mL)	observed concentration  (μg/mL)	SD µg/mL	Precision RSD (%)	Accuracy (%)
DS	Hydrochlorothiazide	12.80	12.89	0.0733	0.569	100.70
		19.20	19.10	0.0894	0.468	99.48
		28.80	28.86	0.0649	0.225	100.21
DS	Triamterene	0.80	0.79	0.0164	2.076	98.75
		6.40	6.38	0.0454	0.712	99.69
		19.20	19.21	0.1854	0.965	100.05

Accuracy (%) = (observed concentration/theoretical concentration) ×100.

 Five separate determinations were performed and calculated the mean.

 

 

 

 

 

Table (4): Recoveries for the four drugs.

          Five separate determinations were performed and calculated the mean.

 

Table 3, a: Results of [(HYD) 35 mg and (TRM) 50 mg]/tab in BIAZID product for five different batches.

No. of batches	HYD 25 mg/tab.				TRM 50 mg/tab.
	Result dose   mg/tab.	SD mg/tab.	RSD %	Per %	Result dose  mg/tab.	SD mg/tab.	RSD %	Per %
1	25.29	0.178	0.704	101.16	51.47	0.431	0.837	102.94
2	25.04	0.447	1.785	100.16	50.79	0.563	1.108	101.58
3	24.74	0.785	3.173	98.96	50.04	0.669	1.337	100.08
4	24.75	0.574	2.319	99.00	49.49	1.008	2.037	98.98
5	25.10	0.356	1.418	100.40	50.51	1.186	2.348	101.02
Range µg/tab	24.74 – 25.29				49.49 – 51.47
Range Per %	98.96 – 101.16				98.98 – 102.94
Range RSD %	0.704 – 3.173				0.837 – 2.348

 Five replicated determinations were performed for every batch.

 

RECOVERY:

The recovery was studied by three addition standards for every product. Table 4 presents the recoveries result for the four Syrian trademark drugs.

 

Method application: Estimation (HYD) and (TRM) in Syrian tablets formulation:

The developed method was applied for quantitative determination for (HYD) and (TRM) in different Syrian tablets formulation. The samples were prepared as described in the section of samples preparation. Quantitative analysis was done by using calibration curves.

 

The obtained results are summarized in Table 3, a-d for four different Syrian trademark drugs. The dosages of (HYD) and (TRM) were conformed to USP legislation. In general, the concentrations of the detected (HYD) and (TRM) in the studied products were within the allowed limits under the Syrian Pharmacopoeia: 12.5, 25 mg/tab for (HYD) and 50 mg/tab for (TRM). The relative standard deviations RSD % (n = 5) of the quantitative results were in the range of 0.704 – 3.173 % and 0.837 – 2.348 % for (HYD) and (TRM), respectively. Table 3, a-d present the determination results of pharmaceutical compounds (HYD) and (TRM), in four Syrian products for five different batches for each: Biazid (HYD) 25 mg + (TRM) 50 mg /tab, Alfares (HYD) 25 mg/tab, Alfares (HYD) 12.5 mg/tab and DIUREX (HYD) 25 mg/tab).

 

Table 3, b: Results of (HYD) 25 mg/tab in Alfares product for five different batches.               

No. of batches	(HYD) 25 mg /tab.
	Result dose mg/tab.	SD mg/tab.	RSD %	Per %
1	24.76	0.437	1.765	99.04
2	24.94	0.417	1.672	99.76
3	24.91	0.536	2.152	99.64
4	25.14	0.451	1.794	100.56
5	24.88	0.370	1.487	99.52
Range µg/tab	24.76 – 25.14
Range Per %	99.04 – 100.56
Range RSD%	1.672 – 2.152

          Five replicated determinations were performed for every batch.

 

 

 

 

 

 

 

Table 3, c: Results of (HYD) 12.5 mg/tab Alfares product for five different batches.

No. of batches	(HYD) 12.5 mg/tab.
	Result dose mg/tab.	SD mg/tab.	RSD %	Per %
1	12.62	0.141	1.117	100.96
2	12.69	0.225	1.773	101.52
3	12.44	0.284	2.283	99.52
4	12.68	0.368	2.902	101.44
5	12.53	0.199	1.588	100.24
Range µg/tab	12.44 – 12.69
Range Per %	99.52 – 101.52
Range RSD%	1.117 – 2.902

         Five replicated determinations were performed for every batch.

 

Table 3, d: Results of (HYD) 25 mg/tab in DIUREX product for five different batches.

No. of batches	(HYD) 25 mg/tab.
	Result dose  mg/tab.	SD mg/tab.	RSD %	Per %
1	24.84	0.263	1.059	99.36
2	24.76	0.319	1.288	99.04
3	24.53	0.224	0.913	98.12
4	24.71	0.331	1.340	98.84
5	24.78	0.248	1.001	99.12
Range µg/tab	24.53 – 24.84
Range Per %	98.12 – 99.36
Range RSD%	0.913 – 1.340

 Five replicated determinations were performed for every batch.

 

According to USP legislation, the tablets must contain not less than 90.00 percent and not more than 110.00 percent of labeled amount. So the obtained results are conformed to USP legislation.

 

CONCLUSION:

Hydrochlorothiazide and Triamterene were estimated individually and in mixtures in different local pharmaceutical products studied in a simple spectral manner. (HYD) and (TRM) using the first derivative spectrum method using the zero-crossing point.

 

The levels of pharmaceutical compounds were within the permissible limits set by the USB legislation¹⁵. The proposed method for estimating (HYD) and (TRM) is accurate, simple, sensitive, straightforward and straightforward in quantitative analysis without prior chemical treatment in an individual or binary mixture.

 

The mixture ratios of (HYD) and (TRM) in pharmaceutical products were different. It was also observed that the level of (TRM) in general was higher than that of (HYD) in all analyzed products.

 

The presence of active substances in actual quantities in pharmaceutical products was conformed to the pharmacological (HYD) and (TRM) formulation.

 

 

ACKNOWLEDGEMENT:

The Ministry of High Education in Syria financially and technically supported this work through department of Chemistry, Faculty of Science, University of Aleppo, Syria.

 

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Accepted on 29.03.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(6): 2351-2356.