Absorbance Correction H-Point Standard Addition Method for Simultaneous Spectrophotometric Determination of Ramipril, Hydrochlorothiazide and Telmisartan in Tablets

 

Lakshmi Sivasubramanian* and K.S. Lakshmi

Dept of Pharmaceutical Analysis, SRM College of Pharmacy, SRM University, Kattankulathur – 603 301, Tamilnadu.

*Corresponding Author E-mail: lakshmiss@hotmail.com

Simultaneous spectrophotometric method described for the determination of Ramipril, Hydrochlorothiazide and Telmisartan in tablets using absorbance correction – H-Point Standard addition method (HPSAM). A simple and novel method absorbance correction – HPSAM is reported for simultaneous estimation of three drugs using UV-visible spectrophotometry without any prior separation of samples. The linear range was 0.8 - 3 µg/mL for Ramipril, Hydrocholrothiazide and Telmisartan. The relative standard deviation (RSD) for the simultaneous determination of 2 µg/mL of Ramipril, hydrochlorothiazide and Telmisartan by applying HPSAM was 0.373, 1.176 and 1.296 respectively. The reported methods HPSAM can be claimed as green analytical chemistry as it does not involve the use of organic solvents and hence can be utilized for the routine analysis of these drugs from tablets.

 

 

INTRODUCTION:

Ramipril, 2- N- (S)-1-ethoxy carbonyl-3-phenyl propyl -lalanyl-(1S,3S,5S)-2-azabicyclo 3,3,0  -octane-3-carboxylic acid is a prodrug  ¹   which is rapidly hydrolyzed with the cleavage of an ester group through hepatic metabolism in human body forming an active metabolite, i.e., Ramiprilat. Hydrochlorothiazide (HCZ), is widely used in antihypertensive pharmaceutical preparations and is chemically known as 6-chloro-3, 4-dihydro-2H-1,2,4-benzothiadiazine-7-sulphonamide 1,1-dioxide^2–4.  Telmisartan (TEL), 4 -  (2-n-propyl-4-methyl-6- (1-methyl benz imidazole-2-yl)-benz imidazole-1-yl) methyl–biphenyl -2-carboxylic acid, is a selective angiotensin II type 1 receptor (AT1R) blocker, which belongs to the group of angiotensin II receptor antagonists⁵  . Figure 1 represents the structure of RAM, HCZ and TEL. A literature survey reveals a variety of spectrophotometric and chromatographic methods such as UV derivative, the simultaneous equation method, colorimetric determination, High Performance Thin Layer Chromatographic method (HPLC), ratio derivative and a stability indicating HPLC methods have been reported for the determination of LOS, AML and HCZ in pharmaceutical dosage forms individually or in combination with other drugs ^6–31.

 

 

 

1

 

 

2

 

3

 

Figure 1: Structure of Ramipril [1], Hydrochlorothiazide [2] and Telmisartan [3]

In this paper the authors report a novel technique called absorbance correction H-point standard addition method (HPSAM). This technique also overcomes the spectral interference without prior separation of components and also gives a general linearity range which can be applied for analysis of samples of any concentration. In 1988, Bosch-Reig and Campins-Falco presented a new technique; the HPSAM based on the principle of dual wavelength spectrophotometry and the standard addition method³² . The greatest advantage of HPSAM is that it can remove errors resulting from the presence of an interfering and blank reagent. However the method faces a drawback of being applicable only for determination of two drugs. Hence the authors propose a new technique – absorbance correction HPSAM to perform quantitative analysis of three overlapping analytes without prior separation.

 

MATERIALS AND METHODS:

Apparatus:

A Perkin Elmer (Lamda 25) Spectrophotometer controlled by UV Winlab software and equipped with a 1 cm pathlength quartz cell was used for UV-Vis spectra acquisition. RAM, HCZ and TEL reference standards were kindly supplied by Madras Pharmaceuticals, Chennai, India. The tablets Telista H (Lupin Pharma, Label Claim – 40 mg TEL and 12.5 mg HCZ), Telma R5 (Glenmark Pharmaceuticals, Label Claim – 40 mg TEL and 5 mg RAM) and Cardace #2.5 (Aventis Pharmaceuticals, Label Claim – 2.5 mg RAM and 12.5 mg HCZ) were procured from local pharmacies. All other chemicals were of analytical reagent grade and procured from SD Fine chemicals, Mumbai, India.

 

EXPERIMENTAL

Standard Solutions:

Standard stock solutions (1000 µg/mL) of RAM, HCZ and TEL were prepared separately in the diluent 0.1M NaOH and water in the ratio 20:80 v/v. These solutions were taken and then diluted to 10 ml with water to give a final analyte concentration desired.

 

Sample Preparation:

Ten tablets of each were weighed and finely powdered in a mortar. An amount  of the powder equivalent to one tablet was accurately weighed and transferred into three separate 100 ml volumetric flask including the diluent. The flask was sonicated for 15 mins and diluted to the mark with diluents. An aliquot of the solution was centrifuged at 5000 rpm for 10 mins. Appropriate amount of clear supernatant was transferred into a 10 mL flask and diluted with water. The absorbance values of these solutions were measured.

 

Calibration of Telmisartan:

The calibration graphs of TEL were constructed in three selected wavelengths 216.32, 228.41 and 295.12 nm and the results are shown in Table 1.

 

Table 1: Linearity results of Telmisartan at selected Wavelengths

TEL - Telmisartan

 

H-Point Standard Addition Method:

An aliquot of the solution containing 10 µg/mL RAM, 20 µg/mL HCZ and 20 µg/mL TEL were added into 10ml volumetric flask and made up to the mark with water. The solution was then allowed to stand for 10 min at room temperature. After that, a portion of the solution was transferred into a quartz cell to measure its absorbance at appropriate wavelengths. Synthetic samples containing different concentration ratios of RAM, HCZ and TEL were prepared and standard additions of RAM up to 20 µg/mL were performed. Simultaneous determination of RAM, HCZ and TEL with absorbance correction and HPSAM was performed (measuring the absorbance at 295.12 nm for direct determination of TEL according to Beer’s law and also calculating the corrected absorbance at 216.32 and 228.41 nm for HPSAM). The linear range for TEL determination is between 2-10 µg/mL in 295.12 nm. The concentration ranges of RAM and HCZ for construction of HPSA calibration graphs was 0.8-3 µg/mL for both the drugs.

 

Requirements for applying HPSAM:

For the simultaneous estimation of three analytes, three wavelengths were selected as l₁, l₂ and l₃. Two wavelengths, l₁, and l₂ were selected from the spectrum of the interferent (Y) at which the absorbance values remains same and the absorbance of the analyte (X) shows a linear response. Third wavelength l₃ was selected from the second interferent (Z), at which both the analyte and the first interferent are free of interferences. The contribution made by the second interferent at l₁, and l₂ is overcome by absorbance correction method as follows:

 

A_{corr, l1} = A_{mix, l1} – r₁ × A_{mix, l3}                    (1)

A_{corr, l2} = A_{mix, l2} – r₂ × A_{mix, l3}                    (2)

 

whereA_{mix, l1}, A_{mix, l2} and A_{mix, l3} are the absorbances of sample (ternary mixture) at l₁, l₂ and l₃ respectively.  A_{corr, l1} and A_{corr, l2} are the net absorbances due to the contribution of second interferent (Z) at l₁ and l₂ respectively, that are used for the construction for H-Point Standard addition graph (HPSA graph). The values r₁ and r₂ are the slope ratios of second interferent, Z calibration graphs:

 

r₁ = Slope in l₁ / Slope in l₃                                     (3)

r₂ = Slope in l₂ / Slope in l₃                                     (4)

 

Wavelength Selection:

To select the appropriate wavelengths for using HPSAM the following principles were applied. At these selected wavelengths the analyte signals must be linear with the concentrations and the interference signal must remain equal, in the case where the analyte concentrations are changed, the analytical signal obtained from the mixture containing the analyte and the interfering should be equal to the sum of the individual signals of the two components. In addition, the difference in the slopes of the two straight lines measured at two selected wavelengths (λ₁ and λ₂) must be as large as possible in order to get good accuracy and sensitivity. In this case there were several pairs of wavelengths. The wavelength pair of 216.32 and 228.41 nm (l₁ and (l₂) was chosen for the study. Standard solutions of RAM and HCZ were initially tested to validate the applicability of the chosen wavelengths.

 

RESULTS AND DISCUSSION:

HPSAM:

In this proposed method, RAM was selected as an analyte and HCZ as an interferent. Several wavelength pairs were examined and the wavelength pair of 216.32 and 228.41 nm was selected. The third wavelength (l₃) was selected at 295.12 nm from the spectrum of TEL at which the other two drugs are free from interference. Under the optimum conditions described above, simultaneous determination of RAM, HCZ and TEL was performed by applying absorbance correction and HPSAM. To check the reproducibility of the method, six replicate measurements of RAM, HCZ and TEL were performed. The concentration of TEL was obtained directly from the absorbance of samples using calibration graph (295.12 nm). After removing the contribution of TEL absorbance from the total absorbance at 216.32 and 228.41 nm, the concentration of interfering component (HCZ) was calculated in each test solution by means of the calibration method using standard solutions and the ordinate value of H-point (A_H). The concentration of RAM was directly obtained (-C_H). The relative standard deviation (RSD) for the simultaneous determination of 2 µg/mL of Ramipril, Hydrochorothiazide and Telmisartan by applying HPSAM was 0.373, 1.176 and 1.296 respectively (Table 3).

 

Accuracy of HPSAM:

For Synthetic Mixture Several synthetic mixtures with different concentration ratio of RAM, HCZ and TEL were analyzed with the proposed method to ensure the accuracy of the method. The results are given in Table 4. The accuracy and precision of the method were found to be satisfactory (Table 2 and 3). Fig 3a and 3b shows the H-Point standard addition plots for several synthetic test solutions. For Pharmaceutical Dosage form In order to check the applicability of the method, the different tablet dosage forms were analysed by the proposed method. The results are shown in Table 4 and found to be accurate.

 

Figure 2: Overlain absorption spectra of RAM, HCZ and TEL

 

Figure 3a  H-Point standard addition plot for the simultaneous determination of Ramipril and Hydrochlorothiazide with constant concentration of Hydrochlorothiazide (2 µg/ml) and different concentrations of Ramipril (1) 3 and (2) 2 µg/ml

 

Figure 3b  H-Point standard addition plot for the simultaneous determination of Ramipril and Hydrochlorothiazide with constant concentration of Ramipril (2 µg/ml) and different concentrations of Hydrochlorothiazide (1) 3 and (2) 2 µg/ml

 

Application:

To evaluate the analytical applicability of the proposed method it was applied to the simultaneous determination of RAM, HCZ and TEL in pharmaceutical preparations containing any two compounds. The results are given in Table 4. The good agreement between the results with the composition values indicated by the suppliers indicates the successful applicability of the proposed methods for simultaneous determination of RAM, HCZ and TEL in pharmaceutical preparations.

 

Table 2: Results of several experiments for the analysis of Ramipril, Hydrochlorothiazide and Telmisartan mixtures at different concentration ratios by absorbance correction – HPSAM

 

RAM – Ramipril, HCZ – Hydrochlorothiazide and TEL – Telmisartan

A – absorbance at respective wavelength, C – Concentration of unknown sample, R² – correlation coefficient

 

 

Table 3: Results for five replicate analysis of Ramipril, Hydrochlorothizide and Telmisartan mixtures by absorbance correction HPSAM

 

RAM - Ramipril, HCZ – Hydrochlorothiazide and TEL – Telmisartan

A – absorbance at respective wavelength, C – Concentration of unknown sample, R² – correlation coefficient

 

Table 4: Simultaneous determination of Ramipril, Hydrochlorothiazide and Telmisartan from formulations using absorbance correction HPSAM

*Mean of six estimations

** Internal Standard Used

 

 

CONCLUSION:

It was observed that RAM, HCZ and TEL in their mixture have the overlapping absorption spectra in the spectral region of 200 and 350 nm. Therefore, the simultaneous spectrophotometric determination of RAM, HCZ and TEL substances in their synthetic and commercial tablets was performed by using Absorbance correction HPSAM. The proposed method does not require prior separation step and spectral derivation for the simultaneous analysis of RAM, HCZ and TEL. Hence the proposed method can be used for the routine analysis and quality control of the marketing tablet formulation containing RAM, HCZ and TEL substances.

 

ACKNOWLEDGEMENT:

The authors thank the Management of SRM University for providing the necessary facilities to carry out this work.

 

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