Quality by Design (QbD) Method for Concurrent Evaluation of Azelnidipine and Telmisartan Using UPLC and Stability Indicating Studies

According to the Quality by Design theory, instead of evaluating analysis based on results, quality should be incorporated into the process. HPLC uses complex mobile phase systems, systems with a high retention period and limited sensitivity, or systems with more than two solvents. Therefore, a QbD strategy has been used to build a new UPLC method. Using the (QbD) Quality by Design technique, the development stage should consider analytical process quality into consideration¹. A QbD is a methodical approach that focuses on products and process expertise, quality risk management, and process control, all backed by reliable research. A QbD is a methodical method of developing a technique that prioritizes quality risk management, product and process expertise, and process control supported by trustworthy research².

The experimental target profile (ATP), as well as key method characteristics (CMA) crucial method attributes, that is, peak area, retention time, symmetric factor, tailing factor, plate count, and resolution between adjacent peaks, are included in the design (DOE)³. Even though many methods based on the QbD principle have been published, there are no definite guidelines for creating QbD-based analytical procedures. Analytical approaches utilized QbD to accomplish various objectives, such as technique creation, optimization robustness studies, etc⁴. Recently, concept of QbD has grown in importance when creating analytical methods using the design of the experiments (DOE). Therefore, a novel UPLC method utilizing a QbD methodology has been developed to estimate Telmisartan and Azelnidipine in tablet dose forms⁵. Telmisartan lowers blood pressure (hypertension). Heart attacks, strokes, and kidney issues are all reduced by lowering blood pressure. The drugs known as angiotensin receptor blockers include Telmisartan (ARBs). It operates by widening blood arteries to facilitate easier blood flow ⁶ Telmisartan has a molecular mass of 514.6 and the chemical formula C₃₃H₃₀N₄O₂. Its melting point ranges from 261^oC to 263^oC. In the pH range of 3 to 9, water is largely insoluble, and strong acids are weakly soluble (except for hydrochloric acid). They are used to treat hypertension alone or in combination with other antihypertensive.

FIGURE: 01 Structure of Telmisartan

The class of drugs known as anti-hypertensive medicines includes Azelnidipine. Azelnidipine blocks Ca2+ from entering the vascular walls of voltage-dependent smooth muscle channels. Channel Ca2+ can be divided into numerous sub-channels of L, T, N, P, Q, and R. Calcium channel L-type promotes the contraction of smooth muscle, which results in high blood pressure. Smooth muscle walls of the vascular smooth muscle relax and lower blood pressure when calcium channels are closed, preventing the smooth muscle from contracting. azetidin-3-yl 3-[1-(di phenyl methyl)]is the molecular name for Azelnidipine.2 methyl-5-propan-2-yl amino -1,4-dihydro-4-nitrophenyl-3,5-dicarboxylate of pyridine. Its chemical composition is C₃₃H₃₄N₄O₆ and its molecular weight of it 582.657. It melts at 193^oC to 195^oC ⁸.

FIGURE: 02 Structure of Azelnidipine

MATERIALS AND METHODS

Software named empower and separation module 2695 is used to merge the data. For sample preparation, tools such as the weighing machine, pH meter, UV Spectrometer, and the Borosil pipettes, burettes, and beakers were employed. Chromatographic condition: Water HPLC was 25 degrees Celsius, autosampler, and a PDA detector. Separation approach: pH 3.0 buffer or phosphate buffer, Inertsil ODS column (4.6*250mm, 5"), Isocratic method 30% ACN, 70% buffer, and a flow rate of one millilitre per minute make up the mobile phase. The utilized wavelength is 225 nanometers. The injection volume is 10-micron litres— 8 minutes total run time.

RESULTS AND DISCUSSION:

Mobile Phase Optimization: Among the early stages of mobile phases studied were Methanol: Phosphate Buffer, Methanol: Ortho Phosphoric Acid Buffer, methanol to acetonitrile of different ratios. Phosphate buffer : acetonitrile (70:30 v/v) is added to mobile phase by maintaing pH 3. Ultraviolet range for Telmisartan 10gram/ml &Azelnidipine 10gram/ml in diluents (components in the mobile phase) was obtained through scanning at 200 to 400-nanometer region. 225 nanometre UV spectrum wavelength was picked.

FIGURE: 03 UV Graph

Phosphate buffer preparation: Take 3.4 grams of potassium dihydrogen phosphate and 1000 millilitres of HPLC water. 0.1M NaOH was added to get the pH until 3. After being sonicated for 10 minutes, through membrane filter 0.45 solution was purified.

Mobile phase preparation: Acetonitrile and phosphate buffer in ratio 30% and 70% are combined and kept in vacuum filtered through 0.45inch then filter and kept in 10 minutes in an ultrasonic water bath.

TABLE: 01 Build Information

TABLE: 02   Factors

TABLE: 03 Responses of drugs

FIGURE: 04 Retention time of Telmisartan                       FIGURE: 05 Tailing factor of Azelnidipine

TABLE: 04 Azelnidipine tailing factor responses

TABLE: 05 Telmisartan and Azelnidipine Area summarized

FIGURE: 06 Overlay Plot

FIGURE: 07 Overlay report

Preparing a Standard Solution: Working standards, properly weigh and add to10ml volumetric flask, add azelnidipine and telmisartan taken in quantities of 8 mg and 40 mg. 7.0 mL of the diluent should be dissolved ultrasonically, and the same solvent should be added. (stock response). From stock solution 0.75ml is taken in to 10ml volumetric flask the desirable concentration will be diluted with the required amount of liquid.

FIGURE: 08 Standard Chromatogram

FIGURE: 09 Telmisartan Chromatogram

FIGURE: 10 Azelnidipine Chromatogram

FIGURE: 11 Precision Chromatogram of Telmisartan and Azelnidipine

TABLE: 06 Precision results Telmisartan and Azelnidipine

Preparation of stock solution: Combine 8 milligrams of Azelnidipine & and 40 milligrams of Telmisartan in a 10-milliliter volumetric flask, using accurate weights as working standards. To get the appropriate volume add 7 millilitres of the diluent and ultrasonically dissolve it, after which you can add more of the same solvent. (Stock reaction).One of the above stocks, solutions, 0.75 ml, should take in 10 ml volumetric flask. Dilute with necessary volume of liquid will be used to get the desired concentration. An RSD of no more than 2% should be present in the region of results from six standard injections.

ACCURACY: Preparation of Standard stock : Take 8mg Azelnidipine & 40mg Telmisartanin in10ml volumetric flask. Reach the necessary volume and add 7 mL diluent and dissolve it. One of the stocks above solutions, 0.75 ml, should be pipette into a volumetric flask that can hold 10 ml of liquid. The required amount of liquid will next be diluted to the desired level.

Make a 50% solution (Regarding the desired Assay concentration):As working standards, precisely weigh 4mg of Azelnidipine and 20 mg of Telmisartan in 10ml volumetric flask.  Approximately take seven millilitres of the solvent once the diluent dissolves to get the volume to the desired value. (Stock reaction). From the above stock solutions, 0.75 ml, should be pipette into a volumetric flask that can hold 10 ml of liquid. The required amount of liquid will next be diluted to the desired concentration.

FIGURE: 12   Accuracy 50% chromatogram of Telmisartan and Azelnidipine

In order to make a 100 % solution (Regarding the desired assay concentration)

Working standard, in 10ml volumetric flask, combine Azelnidipine 8 mg and Telmisartan 40mg. Weigh the mixture carefully. To increase the volume up to level, add 7 mL of the diluent, sonicate it until it is fully dissolved, and then repeat the process. (Stock reaction). From the stock above solutions, 0.75 ml pipette and transfer in 10 ml volumetric flask. The required  liquid should be diluted to the appropriate concentration after that.

FIGURE: 13 Accuracy 100% chromatogram of Telmisartan and Azelnidipine

In order to make a 150 % solution (Regarding the desired assay concentration): Working standard of 12 mg of Azelnidipine and 60 mg of Telmisartan that has been precisely weighed. Once the diluent has been thoroughly dissolved, add about 7 mL of it. In volumetric flask (10ml) ,1.5ml of stock solution is added and dilute to desired concentration.

FIGURE: 14   Accuracy 150% chromatogram of Telmisartan and Azelnidipine

TABLE: 07 Telmisartan accuracy results

TABLE: 08 Azelnidipine accuracy results

Preparation of Level – I: To dilute the stock solution, add 0.25 ml to 10 ml in a volumetric flask..

Preparation of Level – II: In10 ml volumetric flask add 0.5 ml of the stock solution was added.

Preparation of Level – III: By adding 0.75 ml into a volumetric flask with ten million litres of diluent, the stock solution indicated above was diluted to the right concentration.

Preparation of Level – IV: The stock solutions were diluted to the required concentration by addition of 1ml to the volumetric flask (10 ml).

Preparation of Level – V:  Put 1.25 milliliters of the stock solution into a 10-milliliter volumetric flask, then use diluents to adjust the concentration.

Procedure:  After injecting, measure the peak area after inserting it into the chromatographic device at each level, and plot the concentration versus peak area and the correlation coefficient. (X-axis represents concentration, Y-axis represents peak area)

SYSTEM SUITABILITY: In a standard solution, the peaks brought on by Telmisartan and Azelnidipine should not have a tailing factor of more than 2.0. The resolution in a standard solution peaks for Telmisartan and Azelnidipine should be at least 2.

Calculation:       

% Assay=ATAS*WS DS*DT WT*Avg  weightLable Claim*P100*100

Where: AT stands for average sample preparation area counts.

AS is an acronym for average preparation area counts.

Working standard weight is WS (as measured in milligrams).

P is the percentage standard purity.

TABLE: 09 Linearity results of Telmisartan

FIGURE: 15 Linearity of Telmisartan

ASSAY RESULTS:(Telmisartan)

191907191265*2010*1.510*100254*101.5*254.320*99.8100*100=100.25%

CALCULATION: (For Azelnidipine)

% Assay=ATAS*WSDS*DTWT*Average weightLabel Claim*P100*100
Where; AT is average area counts.

AS is an acronym for average preparation area counts.                  

Label Claim = mg/ml.Working standard weight is WS (as measured in milligrams).   

P is the percentage of working standard purity. 

TABLE: 10 Linearity results of Azelnidipine

FIGURE: 16 Linearity of Azelnidipine

Results for System Suitability:

1. The standard injection's tailing factor was 1.11.
2. The standard injection yields 2381.56 theoretical plates.
3. The standard injection yields a resolution of 4.42.

ASSAY RESULTS: (For Azelnidipine)

106240107234*8.1910*1.510*10254*101.5*254.38.1*99.8100*100=98.99%

The correlation coefficient must be at least 0.999 in order to be accepted.

DETECTION LIMIT: LOD:

Prep of 300µg/ml sol: To completely dissolve the 40 mg of Telmisartan working standard, it must be sonicated and added to around 7 mL of diluent after being precisely measured and poured in a dry, clean, 10-milliliter volumetric flask. The volume is increased to sufficient level, add more liquid while using the same solvent. 0.75 ml of stock solutions in 10ml volumetric flask increasing the concentration as appropriate. S/N The LOD solution must have a ratio value of 3.

LIMIT OF DETECTION: (Azelnidipine)

Preparation for 60 microgram/ml solution:  Add 7 mL of diluent and 8 mg of Azelnidipine working standard accurately measuring and transfer in volumetric flask (10ml). Continue adding liquid the solvent until the required volume is obtained. Adjust the concentration as appropriate and transfer 0.75 ml of the stock solutions in volumetric flask (10ml) S/N the LOD solution must have a ratio of three.

FIGURE: 18 LOD of Telmisartan and Azelnidipine

LIMIT OF QUANTIFICATION: (Telmisartan)

Preparation of 300 g/ml sol:  To completely dissolve 40 mg of Telmisartan working standard, it must be sonicated and added to around 7 mL of diluent after being accurately measured and transfer in 10ml volumetric flask capacity.  Volume can be adjusted to the required level. Take 0.75 ml of stock solution in 10ml volumetric flask, add diluents to adjust the concentration (stock reaction). S/N ratio value for LOQ must be 10.

(LOQ) LIMIT OF QUANTIFICATION: (Azelnidipine)

60 µg/ml solution preparation: In 10 ml volumetric flask, 8 mg of Azelnidipine working standard should be properly measured, and sonicated to completely dissolve the diluent. Continue adding liquid using the same solvent until the required volume is obtained. (Stock reaction)

Preparation for 5.64 µg/ml solution: Fill a 10ml volumetric flask with 0.94 ml of the stock solution. After that dilute with enough liquid to get the required concentration. For the LOQ solution, the S/N Ratio needs to be 10.

FIGURE: 19 LOQ of Telmisartan and Azelnidipine

ROBUSTNESS: A. Flow rate ranged between 0.9 and 1.1 ml/minutes. Response in addition to the process flow rate, a variety of flow rates were used for the preparation and analysis of 300 ppm of Telmisartan and 60 ppm of Azelnidipine. The fact that the approach still yields accurate results when the flow rate varies by 10% is thus demonstrated

B. Organic composition in the Mobile phase was ±10%.Both mobile phase composition and  modified mobile phase composition were used in this method to produce and analyse 300ppm  Telmisartan and 60 ppm Azelnidipine. According to aforementioned data, the variation is 10 percent.

TABLE: 11 Telmisartan system suitability results

TABLE: 12 Azelnidipine system suitability results

TABLE: 13 Telmisartan system suitability results

TABLE: 14 Azelnidipine system suitability results

According to ICH "stability testing of novel pharmaceutical substances, Stress testing needs to be done to determine component's innate stability characteristics. This research aimed to test if Telmisartan and Azelnidipine would degrade under stress using the provided methods.

Preparation of stock: Use exact weights to weigh and transfer Azelnidipine 8 mg and Telmisartan 40 mg into a 10 ml volumetric flask as working standards. Addition of 7 mL of the diluent, ultrasonically dissolve it to get the right volume, and then add more of stock solution.

Degradation of acid condition:3ml of 0.1N HCl & 0.75 ml the previously described combination should be added with a pipette in 10 ml volumetric flask and neutralized with 10ml of 0.1 N NaOH .

FIGURE: 20 Acid Degradation of Telmisartan and  Azelnidipine

Degradation of alkaline condition: In a 10 ml volumetric flask, add 3 ml of 0.1N NaOH to 0.75 ml of stock solution.Pour the filtered sol in vials by using syringe filters with 0.44-micron pore size. Telmisartan, Azelnidipine are taken in Petri plate for 3 hours at 110⁰ C. The sample is diluted using diluents, and then subjected to UPLC analysis.

FIGURE: 21 Base Degradation of Telmisartan and Azelnidipine

Oxidative degradation: To the volumetric flask (10 ml), 0.75 ml of the stock solution and 1 ml of 30% H₂O₂ are added. After that, the vol flask kept at room temp for 15 minutes.  filtrated solution after passing it through 0.45-micron syringe filters.

FIGURE: 22 Peroxide Degradation of Telmisartan and Azelnidipine

TABLE: 15 Showing the Results of % Degradation of Telmisartan&Azelnidipine