INTRODUCTION:
Telmisartan (TEL) chemically 4’ {[4-Methyl-6-(1-Methyl-1H-benzimidazol-1-yl)-2propyl-1H benzimidazol-1-yl] Methyl}-2-biphenyl carboxylic acid is an angiotensin II receptor antagonist with actions similar to those of losartan. It is used in the management of hypertension and for the prophylaxis of cardiovascular events1-3.
For estimation of TEL methods such as HPLC5,6, titrimetric method7, and Spectrophotometric method8-10 have been reported for the estimation of TEL alone or in combination with other drugs.
Amlodipine besylate (AMD), 2 - [(2 - amino ethoxy) - methyl] - 4 - (2 - chloro phenyl) -1, 4 -dihydro - 6 - methyl - 3, 5 - pyridine dicarboxylic acid 3 - ethyl - 5 - methyl ester benzene sulfonate, is a potent dihydro calcium channel blocker1-4. Various analytical methods have been reported for the assay of AD alone or in combination with other anti - hypertensive agents in pharmaceutical dosage form include RP HPLC11,12, Chemo metric assisted spectroscopic method13 and UV spectroscopic methods14-20.
Both drugs are official in recently published British Pharmacopoeia21 and Indian Pharmacopoeia22. Chemical structure of both drugs is shown in (Fig No 1).
Fig No 1: Chemical structure of Drug molecule
Quality by design concept is applied for the development of pharmaceutical processes to assure a predefined product quality. QBD concepts are mentioned in ICH guidelines Q8 (R1) (Pharmaceutical development), Q9 (Quality risk management), and Q10 (Pharmaceutical quality system)23-25. ICH guidelines Q8 (R2) defines QBD as a “a systematic approach to development that begins with predefined objectives and emphasizes product and process understanding and process control, based on sound science and quality risk management”26. QBD approach in analytical method summarizes a complete understanding of how the analytical technique attributes and operating conditions affect the analytical performance27,28. Factors to study in analytical quality by design (AQbD) approach may include the type of analytical technique chosen; reagents used and instrument parameters29,30.
There are similar advantages of applying QbD principles to analytical methods as to manufacturing processes and product31. A QbD approach can be beneficial in the development of suitable, robust, low cost and eco-friendly (eco-friendly solvent, chemicals) method which is applicable at any stage of the lifecycle of the product. Also some regulatory guidelines have mentioned flexibility of changing analytical method without revalidation if the AQbD approach has been implemented during analytical method development. The first stage of AQbD approach is to fix an analytical target profile (ATP) for the method. ATP defines the goal of the analytical method development process and it is the sign of method performance32,33. For analytical method validation ICH Q2 (R1) has given various method performance characteristics for an analytical method. Thus a QbD based UV spectrophotometric was developed, QbD approach was implemented with the study of the effect of method input variables on spectral shape, intensity of absorbance, and absorbance maxima λmax and critical parameters were selected for the proposed method and method was validated as per ICH guidelines Q2 (R1).
MATERIALS AND METHODS:
Instrumentation:
Analysis was performed with a Shimadzu Double beam UV-Visible spectrophotometer (Shimadzu, Kyoto, Japan) with spectral bandwidth of 2nm and wavelength accuracy of ± 1nm with 10mm matched Quartz cells was used. Electronic balance Afcoset balance (The Bombay Burmah Trading corpo Ltd) with accuracy ±0.1mg Model No. ER 200A was used for weighing and for degassing the solution Digital Ultrasonic cleaner 1.8Ltr (Labman scientific Instruments Chennai) was used.
Reagents and Chemicals:
Pharmaceutically pure samples of AMD from Smruthi Organics Ltd Solapur Maharashtra and TEL from Swapnroop Drugs and Pharmaceuticals, Aurangabad Maharashtra were procured as a gift sample and the commercial formulation containing Telmisartan 40mg and Amlodipine 5mg was procured from the local market.
AQbD approach application in method development:
AQbD approach was applied to study the influence of input variable parameters on spectrophotometric analytical method performance shown in (Fig No 2).
Solvent selection:
AMD is freely soluble in methanol, slightly soluble in water and isopropyl alcohol, whereas TEL is sparingly soluble in alcohol, dichloromethane, slightly soluble in methanol and practically insoluble in water. Although the solubility of the procured drugs was studied in methanol, 0.1 N HCl and 0.1 N NaOH, 0.05 N HCl and 0.05 N NaOH separately; and each solution with known conc of analyte were scanned in UV range of 210 nm to 400nm. The recorded overlain spectra in respective solvent are shown in Fig No 3, 4, 5 and 6. It was found that suitable solvent is 0.1 N HCl with respect to low cost, robust and precise in producing result.
Fig No 2: Diagram showing the relationship between input variable parameters and the spectrophotometric method performance characteristics
Fig. 3: Overlain spectrum of both drugs in 0.1 NaOH
Fig. 4: Overlain spectrum of both drugs in 0.1 N HCI
Fig.5: Overlain spectrum of both drugs in 0.1 N HCI
Fig. 6: Overlain spectrum of both drugs in methanol
Preparation of stock solutions and standard solutions:
10mg each of drug TEL and AMD were separately and accurately weighed; and transferred into separate 50ml volumetric flask. Dissolved into 0.1 N HCl and volume was made to 50ml with solvent. Subsequent standard solution of each drug with conc 10mcg/ml was prepared by diluting aliquot 0.5ml of stock solution to 10ml capacity volumetric flask.
Selection of wavelength and conc range:
From UV spectra it was found that TEL has measurable absorbance at 226nm and 291.2nm; and AMD has maximum absorbance at 237nm and 365.2nm. At 365.2 nm TEL was zero absorbance hence this wavelength was suitable for measurement of AMD; and 291.2nm where AMD has poor absorbance so thoughtfully applied absorbance correction method for measurement of TEL. Chemometric method was applied and which was reasonable remedy to overcome interference of AMD. From the nature of spectra working conc range 5 to 40 mcg/ml for AMD and 5 to 40mcg/ml for TEL was selected in 0.1 N HCl. Also combined drug solution was prepared simulated to marketed formulation. Selected critical parameters based upon above discussion, observations were listed in Table No 1 and by using these; method was validated as per ICH guidelines and by analysing marketed preparations.
Table No 1: Selected critical parameters for UV-VIS analytical method of AMD and TEL
|
Parameter |
Selected variables of method I AMD TEL |
Selected variables of method II AMD TEL |
||
|
Wavelength |
365.2 |
291.2 |
365.2 |
291.2 |
|
Solvent |
0.1 N HCl |
0.1 N HCl |
0.1 N HCl |
0.1 N HCl |
|
Scan speed |
Fast |
Fast |
Fast |
Fast |
|
Sampling interval |
0.2 nm |
0.2 nm |
0.2 nm |
0.2 nm |
Experimental Method for estimation:
From the overlain spectra two methods absorbance correction method and multicomponent method were applied for simultaneous estimation of both drugs in the combined dosage form.
Method-I Absorbance correction method:
TEL was shown absorbance at 226nm and 291.2nm; and AMD has maximum absorbance at 237 and 365.2nm. At 365.2nm zero absorbance of TEL was found hence this wavelength was suitable for exclusive measurement of AMD; and 291.2nm where AMD shows interference, which was corrected by absorbance correction. The equation A= abc was applied for x (AMD) and y (TEL) determination. Working standard solutions of AMD and TEL containing 10mcg/ml conc were separately prepared and used for the method.
At 365.2nm A = ax2 .b.cx
Where A = absorbance of AMD sample
ax2 = absorptivity of drug at 365.2 nm
b = Pathlength of solution 1 cm
cx = Conc of AMD in sample
At 291.2 nm A s = A1 + A2
A s = aX1 .b. Cx + ay1 .b. Cy
On rearranging equation
As = absorbance of sample containing AMD and TEL at 291.2 nm
aX1 = absorptivity of AMD at 291.2 nm
ay1 = absorptivity of TEL at 291.2 nm
b = Pathlength of solution 1 cm
cy = Conc of TEL in sample
Method- II Multicomponent method:
In interpretation of spectra it was observed that both drugs has less interference at λmax by another one, hence wavelength 291.2nm and 365.2nm were selected. The characteristics of spectra was guided that the use of mixed standards were found appropriate than the use of pure standards. Mixed standards containing different proportions of both the drugs were rationally experimented keeping in view the conc.of the drugs in the available formulations. Six mixed standards were selected for quantitative analysis shown in Table No 2.
Table No 2: Mixed standard solutions containing TEL and AMD in mcg/ml
|
Standard No |
1 |
2 |
3 |
4 |
5 |
6 |
|
Conc of TEL in mcg/ml |
0 |
5 |
10 |
20 |
30 |
40 |
|
Conc of AMD in mcg/ml |
40 |
30 |
20 |
10 |
5 |
0 |
Sample solutions were prepared in the TEL: AMD ratio of 1: 1, 1: 2, 1: 3, 2: 1, 3: 1 and sampling wavelength and conc. of each drug in the six mixed standards were provided to the instrument using multicomponent mode of the instrument. Subsequently all the mixed standards were scanned in the range of 400 to 210nm. The instrument was collected and computed spectral data from the mixed standards employing matrix equations; and used for quantitative analysis of the samples. The conc of each of the drug in the sample solutions were computed and printed out by the instrument.
Validation of the Method:
Selected critical parameters should meet the performance characteristics of the analytical method so as to attain analytical target profile of the method. An ICH guideline Q2 R1 was applied to study methods performance with critical parameters in order to implement AQbD approach. The method was validated as per ICH guidelines
System suitability:
System suitability is studied to demonstrate the suitability of the developed procedure under consideration for the analytical method. Six replicates of working standard solutions with conc 10mcg/ml each of AMD and TEL were prepared separately and absorbance was recorded, and SD and %RSD of the response was calculated.
Linearity:
The linearity of an analytical method is its ability to obtain response i.e. absorbance which is directly proportional to the conc of analyte. series of working standard solutions were prepared in conc. range of 5 to 40mcg/ml for AMD and 5 to 40mcg/ml for TEL and scanned in 210 to 400nm range in spectrum mode of the spectrophotometer, absorbance of the standard solutions were recorded at 291.2 for TEL and 365.2nm for AMD in spectrum order. Microsoft office excel software tool was used to obtain the standard regression curve and its analysis as slope, intercept, and correlation coefficient.
Assay of formulation:
Assay was carried out by proposed methods and assay was validated by statistical parameters.
Estimation of formulations by absorbance correction method:
Tablet powder equivalent to 2.5mg AMD and 20mg TEL was weighed and transferred into 50ml volumetric flask. Dissolved into 0.1 N HCl and volume was made to 50ml with solvent. Solution was filtered through whatman filter paper and aliquot of solution was further diluted to obtain sample. Solution was scanned in the range of 210 to 400nm to obtain absorbance and record at 291.2 and 365.2nm in spectrum order. Obtained absorbance was utilised to estimate unknown conc of formulation; and results were statistically validated to obtain % of nominal conc, standard deviation and % of RSD.
Estimation of formulations by Multicomponent method:
Above prepared tablet Solution was scanned in 210 to 400nm range and conc of each of the drug component in the tablet solution was obtained in Multicomponent method. Obtained results are statistically validated to obtain % of nominal conc, standard deviation and % of RSD.
Accuracy and Precision:
The accuracy of an analytical method expresses the closeness of an agreement between test result and true result. Accuracy study was performed by recovery study i.e. standard addition method; diluted standard solutions of AMD and TEL were prepared and standard solutions added in 80,100 and 120% proportionate to the tablet solution. Three replicates at each of these three levels were prepared, measured and % of conc, SD and RSD of replicates were calculated.
The precision study was carried out by performing assay of tablet six times; also the reproducibility in result was ascertained by interday and intraday precision.
Limit of Detection (LOD) and Limit of Quantitation (LOQ):
The LOD and LOQ of AMD and TEL by the proposed method were determined using calibration graph method and calculated as 3.3σ/s and 10 σ/s for LOD and LOQ respectively, where σ is the standard deviation of calibration curve and s is the slope of regression line.
Robustness and Ruggedness:
It is measure of capacity of analytical procedure to remain unaffected by small but deliberate variations in method parameter.
RESULTS AND DISCUSSION:
Method development comprises numerous steps, of which solvent selection, method for measurement selection are significant one. Uses of aqueous solvents, eco-friendly solvents like hydrotropic have got remarkable weightage due to low cost, readily available and environmentally sound. Drugs underlying analysis must have appreciable solubility in the selected solvent. Chemical structure of the drug and physico-chemical properties available in the literature guides about use of appropriate solvent in the method.
System Suitability:
The absorbances of six replicates of standard solutions (10mcg/ml) are reported in Table No 3. The SD and % RSD was found for AMD and TEL and meets the system suitability requirements indicates method was suitable for analysis.
Table No 3: System suitability study of AMD and TEL
|
Sr No |
Conc in mcg/ml |
Absorbance of AMD |
Absorbance of TEL |
|
1 |
10 mcg/ml |
0.123 |
0.596 |
|
2 |
10 mcg/ml |
0.119 |
0.605 |
|
3 |
10 mcg/ml |
0.116 |
0.612 |
|
4 |
10 mcg/ml |
0.115 |
0.598 |
|
5 |
10 mcg/ml |
0.116 |
0.618 |
|
6 |
10 mcg/ml |
0.112 |
0.620 |
|
|
SD RSD |
0.003535 2.97051 |
0.010127 1.68914 |
Linearity:
The calibration curve of both drugs was found to be linear in the conc range of 5-40mcg/ml for AMD and 5-40mcg/ml for TEL as shown in Fig No 7. The regression equation of line and its parameters slope, r2 value and intercept are tabulated in Table No 4, which proved the linear relationship between conc and obtained response.
Fig No 7: Calibration curve of TEL and AMD in 0.1 N HCl solvent
Table No 4: Parameters of regression equation obtained in Microsoft excel
|
Parameters |
TEL |
AMD |
|
Detection wavelength |
291.2 |
365.2 |
|
Beer’s law limit (μg/ml) |
5 – 40 mcg/ml |
5 – 40 mcg/ml |
|
Correlation coefficient (r2) |
0.9998 |
0.9993 |
|
Regression equation (y = mx + c) |
Y = 0.0059 X + 0.0139 |
Y = 0.0104 X – 0.0035 |
Assay:
The assay was carried out by both the methods. The spectra of formulation by method I was shown in Fig No 8 and the overlain spectra obtained in method II was shown in Fig No 9. The assay of formulation was carried out by proposed method and calculated % of nominal conc and RSD was found within acceptable limits are summarized in Table No 5. The results indicated applicability of the method for estimation of formulation.