DEVELOPMENT OF HPLC STABILITY DEMONSTRATING METHODOLOGY FOR QUANTIFYING AZELNIDIPINE AND TELMISARTAN IN TABLETS AND BULK TYPES: VALIDATION FOLLOWING ICH DIRECTIVES

Objective: Azelnidipine (AZEL) and Telmisartan (TELM) combination is referred to the sufferers of hypertension. No analytical process has yet been mentioned for the TELM and AZEL combination analysis. We, therefore, have designed for its first time stability demonstrating methodology based on HPLC for analysing TELM and AZEL in the tablets and bulk. Methods: The assay of TELM and AZEL was get done on a 250 mm length C18 column (Supelco, 4.6 mm inner diameter, 5.0 μm particle size) , and utilized 0.1M Na 2 SO 4 (pH 3.6) and acetonitrile (55 % volume: 45 % volume) as the mobile solvents phase, at a stream rate 1.0 ml/min. HPLC recognition of TELM and AZEL was taken by a photodiode array sensor set at 258 nm. For validation of the stability demonstrating methodology proposed in terms of sensitivity, precision, specificity, linearity, device adequacy, robustness and accuracy, ICH directives were followed. Results: Calibration curves of TELM and AZEL were generated in the array of 20- 60 µg/ml and 4 - 12 µg/ml with recovery percentage ranges of 99.62% - 101.05% and 97.76% - 100.17% , and detection limits of 0.020 µg/ml and 0.009 µg/ml , respectively. TELM and AZEL stability was inspected in the existence of acid, base, light, heat, and oxidation and it was realised to be more stable under oxidation degradation testing conditions employed when compared to acid, alkaline, photo, and heat degradation testing conditions applied. Conclusion: The observations demonstrated that the described HPLC stability demonstrating methodology was suitable for quantitating TELM and AZEL combination in tablets and bulk.


INTRODUCTION
Hypertension is the extremely avoidable contributing factor for cardiovascular impairments namely coronary cardiac disease, heart attack, heart stroke, atrial fibrillation, pulmonary artery disease and myocardial infarction, progressive renal disease and cognitive decline [1]. Hypertension is the foremost single contributor to the world's leading basis of demise and disability. In 2010 it is valued that 1.39 billion (31.1%) of adults globally be ill with hypertension. In middle and small revenue nations (1.04 billion people and 31.5%), the occurrence of hypertension among adults was elevated than in the high revenue nations (349 million individuals and 28.5%) [2].
Azelnidipine (AZEL), structure in fig. 1, is a modern dihydropyridine calcium passage antagonist that is particular for the L-kind calcium passages and has received FDA authorization for therapy of the hypertension clients [3]. Antihypertensive outcomes of AZEL are equivalent to that of amlodipine [4]. AZEL is indeed increasingly lipid-soluble and has greater selectivity for the vascular surface than older generational calcium passage antagonists, and in animal experiments treated with AZEL, blood flowing to the brain was markedly enhanced [5].
Telmisartan (TELM), structure in fig. 1, is an angiotensin (II) receptor blockade being used to alleviate minimal to severe hypertension [6][7][8]. TELM is extremely selective for the type 1 angiotensin (II) receptors. TELM is an extra lipophilic angiotensin (II) receptor blocker than utmost other angiotensin (II) receptor inhibitors, which aids its oral uptake and tissue as well as cell permeation [9]. TELM too has an impact on the peroxisome proliferator triggered receptors, which are nuclear hormone receptor superfamily ligand triggered transcription elements. TELM, a drug that treats both diabetes and hypertension, may well be an alternative therapy choice [10,11].
The combination of TELM and AZEL is recommended for hypertension sufferers. In hypertension sufferers, TELM and AZEL combination drops blood pressure, enables blood to flow more effectively to numerous tissues, allows the heart to operate more effectively, and increases oxygen flow across the body, and thereby reducing heart-associated chest pain [12].

Fig. 1: Azelnidipine (AZEL) and Telmisartan (TELM) structures
Stress tests must be implemented on a medication compound to ascertain its basic stability properties, bestowing to the "ICH Q1A (R2)" parent drug permanency evaluation recommendations [13]. Understanding how a drug substance's consistency varies with times and the nature of deterioration products generated under different storage circumstances affects the potency and wellbeing of pharmaceuticals is critical [14][15][16]. Stability demonstrating methods are a group of analytical methodologies that demonstrate the sample stability and must be completely validated. No Stability demonstrating method has yet been recommended for the TELM and AZEL combination. We developed, for the initial time, stability demonstrating methodology for TELM and AZEL combination in tablets and bulk in this report. The approach developed is indeed dependent on reverse-phase liquid chromatography with a photodiode sensor, that has been configured for the investigation of the TELM and AZEL combination in a fast, precise, and sensitive manner.

Instrumentation and analysing conditions of TELM and AZEL combination
High-performance liquid chromatography (Waters 2695 model HPLC system, Autosampler) accompanying with photodiode detector (2998 model) was used in the procedure development and combination analysis of TELM and AZEL. The very fine particles loaded 250 mm length C18 column (Supelco, 4.6 mm inner diameter, 5.0 μm particle size) was utilised for TELM and AZEL chromatographic separation. Flow of 1 ml/min, utilised for the TELM and AZEL chromatographic separation. The mobile phase components of 0.1M Na2SO4 (pH 3.6) and acetonitrile (55% volume: 45% volume) was used. The complete process was completed at room temperature. To every sample, the specimen (10 µl) was chosen as the injection quantity. The quantitation of TELM and AZEL was done at 258 nm.

Chemicals
"SD Fine chem Ltd (India)" supplied analytical mark Na2SO4, peroxide and NaOH; "Merck (India)" supplied analytical mark HCl, H3PO4 and HPLC mark acetonitrile; Milli Q System base prepared Milli Q water was employed in the combined analysis of TELM and AZEL.

Reference drugs and tablets
"Rainbow pharma training laboratories (India)" provided TELM and AZEL reference bulk samples. TELMA-AZ tablet type (Glenmark Pharmaceutical Ltd, India) was bought from a local shop drug retailer and claimed to comprise 8 mg of AZEL and 40 mg of TELM.

TELM and AZEL combination solutions
A stock TELM and AZEL combination solution (concentration: 400 µg/ml TELM and 80 µg/ml AZEL) was made by solubilizing 40 mg of TELM and 8 mg of AZEL in 25 ml of mobile phase and then diluting to 100 ml volume with the very similar solvent. A working TELM and AZEL combination solution (concentration: 40 µg/ml TELM and 8 µg/ml AZEL) was made by solubilizing 1 ml of stock TELM and AZEL combination solution in 3 ml of mobile phase and then diluting to 10 ml volume with the very similar solvent.

Procedure for evaluating the TELM and AZEL combination in bulk
Different aliquots covering 20 μg/ml-60 μg/ml of TELM and 4.0 μg/ml-12.0 μg/ml of AZEL were correctly shifted from stock TELM and AZEL combination solution (concentration: 400 µg/ml TELM and 80 µg/ml AZEL) into separate sets of 10 ml volumetric flasks, and then diluting to 10 ml volume with the very similar solvent. 10 µl volume of each solution were infused into 250 mm length C18 column (Supelco, 4.6 mm inner diameter, 5.0 μm particle size) and evaluated with "stability-indicating HPLC methodology" conditions given (see section: Instrumentation and analysing conditions of TELM and AZEL combination). The peak response values of TELM and AZEL at 258 nm were recorded and afterward calibration curves of TELM and AZEL were drawn followed by calculating regression equations of TELM and AZEL. The concentration of TELM and AZEL in nameless specimen solution can be assessed by exploiting the calibration curves of TELM and AZEL or regression equations of TELM and AZEL, respectively.

Procedure for evaluating the TELM and AZEL combination in tablets
A total of 20 tablets (TELMA-AZ tablet, claimed to comprise 8 mg of AZEL and 40 mg of TELM) were collected, with the median weight estimated and mashed to a fine powder. Dose comparable to 40 mg of TELM and 8 mg of AZEL was shifted to a volumetric flask (100 ml), blended with 10 ml of mobile and stirred with ultra sonicator at 27 °C for 20 min, diluted with similar solvent up to 100 ml mark and filtered via a filter membrane (0.45 m). This tablet stock solution has 400 µg/ml TELM and 80 µg/ml AZEL. A sample tablet TELM and AZEL combination solution (threotical concentration: 40 µg/ml TELM and 8 µg/ml AZEL) for the analysis was made by solubilizing 1 ml of tablet stock TELM and AZEL combination solution in 3 ml of mobile phase and then diluting to 10 ml volume with the very similar solvent. 10 µl volume of sample tablet TELM and AZEL combination solution were infused into 250 mm length C18 column (Supelco, 4.6 mm inner diameter, 5.0 μm particle size) and evaluated with "stability-indicating HPLC methodology" conditions specified (see section: Instrumentation and analysing conditions of TELM and AZEL combination). At 258 nm, the peak response values of TELM and AZEL were measured. The amount of TELM and AZEL in the tablet specimen solution can be established by employing TELM and AZEL calibration curves or regression equations, respectively.

Stability testing of TELM and AZEL
The stability of TELM and AZEL should be assessed by a stress testing stability analysis. This research illustrates the stability of TELM and AZEL in the presence of acid, base, light, heat, and oxidation [13].

Acid degradation testing
The investigation was done out using 0.1N hydrochloric acid (10 ml). The TELM and AZEL stock tablet solution (400 µg/ml TELM and 80 µg/ml AZEL) was combined in an equivalent volume proportion with acid and blended into the volumetric flask (100 ml). The blend was stirred for 30 min at 27 °C using a ultra sonicator. The sample was mixed with mobile phase up to 100 ml mark and filtered via a filter membrane (0.45 m) before being inserted (10 µl) into the HPLC instrument for TELM and AZEL analysis with "stabilityindicating HPLC methodology" conditions specified (see section: Instrumentation and analysing conditions of TELM and AZEL combination).

Oxidation degradation testing
The oxidation degradation testing of TELM and AZEL was worked out with peroxide (30%, 10 ml) as the oxidising agent. In a similar quantity volume, the stock TELM and AZEL tablet solution (400 µg/ml TELM and 80 µg/ml AZEL) is blended with peroxide. The sample was stirred with ultra sonicator at 27 °C for 30 min, diluted with mobile phase up to 100 ml mark and filtered via a filter membrane (0.45 m). The sample (10 µl) was inserted into HPLC machine and the TELM and AZEL content were established with "stability-indicating HPLC methodology" conditions specified (see section: Instrumentation and analysing conditions of TELM and AZEL combination).

Alkaline degradation testing
The investigation was done out using 0.1N sodium hydroxide (10 ml). The tablet TELM and AZEL stock solution (400 µg/ml TELM and 80 µg/ml AZEL) was combined in an equivalent volume proportion with alkaline and blended into the volumetric flask (100 ml). The blend was stirred for 30 min at 27 °C using a ultra sonicator. The sample was mixed with mobile phase up to 100 ml mark and filtered via a filter membrane (0.45 m) before being inserted into the HPLC instrument for TELM and AZEL analysis with "stability-indicating HPLC methodology" conditions specified (see section: Instrumentation and analysing conditions of TELM and AZEL combination).

Photodegradation testing
For this investigation, the stock solution of TELM (400 µg/ml) and AZEL (80 µg/ml) was placed into the volumetric flask (100 ml) and exposed with sunlight for 6 h. After that, the sample was mixed with mobile phase up to 100 ml mark, filtered via a filter membrane (0.45 m) and analysed for TELM and AZEL content using "stabilityindicating HPLC methodology" conditions specified (see section: Instrumentation and analysing conditions of TELM and AZEL combination).

Heat degradation testing
For this report, the stock solution of TELM (400 µg/ml) and AZEL (80 µg/ml) was placed into the volumetric flask (100 ml) and exposed with 60 °C for 30 h using an oven. After that, the sample was mixed with mobile phase up to 100 ml mark, filtered via a filter membrane (0.45 m) and analysed TELM and AZEL content using "stability-indicating HPLC methodology" conditions specified (see section: Instrumentation and analysing conditions of TELM and AZEL combination).

Optimizing the analysing conditions of TELM and AZEL combination
With a "Supelco C18 column (5 μm; 4.6 × 250 mm)" with a column slit temperature having 25 °C and 0.1M Na2SO4-acetonitrile (pH 3.6 and ratio 55:45, v/v) as mobile solvents phase and an isocratic stream form run of 1.0 ml/min, optimal response, good system fittingness values (table 1) and proportioned peak nature for TELM and AZEL were obtained. The best match for the finest peak response and to quantify TELM and AZEL was recognized to be UV identification with a 258 nm configuration. Chromatographic analysis run phase for the TELM and AZEL evaluation was 7.0 min with retaining times of 2.757 and 3.664 min perceived for TELM and AZEL, respectively ( fig. 2).

Quantification and detection limits for TELM and AZEL
The detection limits were characterized as the amount (µg/ml) of TELM and AZEL that should have being sensed and will yield signal/noise proportion of 3:1. The calculated detection limits for TELM and AZEL were 0.020 µg/ml and 0.009 µg/ml, respectively. The quantitation limits were characterized as the amount (µg/ml) of TELM and AZEL that should have being sensed and will yield signal/noise proportion of 10:1. The calculated quantification limits for TELM and AZEL were 0.065 µg/ml and 0.031 µg/ml, respectively. The TELM and AZEL chromatograms at their quantification and detection limit concentrations were represented in fig. 5.

Precision and accuracy
These two criteria for HPLC based stability demonstrating approach was weighed up by the analysis of standard TELM and AZEL solution (40 µg/ml TELM and 8 µg/ml AZEL) by six repeated evaluation of two pure samples of TELM and AZEL on one day, the calculations resulted were exemplified in table 2.

Recovery
This criterion for the HPLC-based stability demonstrating approach was weighed up employing the standard adding methodology. The formulation solutions (40 µg/ml TELM and 8 µg/ml AZEL) were prepared and spiked at quantities comprising 19.8 µg/ml TELM and 3.96 µg/ml AZEL (50% standard adding level), 39.6 µg/ml TELM and 7.92 µg/ml AZEL (100% standard adding level) and 59.4 µg/ml TELM and 11.88 µg/ml AZEL (150% standard adding level). These formulation solutions were assessed by three repeated evaluations, the calculations resulted were exemplified in table 3.

TELM and AZEL stability testing
The stability of TELM and AZEL in the presence of acid, base, light, heat, and oxidation was illustrated with the formulation solution.
The TELM and AZEL chromatogram after acid degradation testing showed four additional peaks at 1.908 min, 5.569 min, 5.835 min and 6.422 min except than TELM peak (2.751 min) and AZEL peak (3.656 min). After acid degradation testing, 10.74% of TELM was degraded and 8.8% of AZEL was degraded. But for the TELM peak (2.752 min) and AZEL peak (6.079 min), the TELM and AZEL chromatograms after alkaline degradation testing revealed four additional peaks at 1.

DISCUSSION
The intention of the present work was to create HPLC-based stability demonstrating approach proficient in separating and determining TELM and AZEL well in the petite feasible analysis time with sensible precision, robust, selective and reliability. The column and solvents of the mobile phase were standardized till a best possible responses and peak structures for TELM and AZEL were achieved [19]. The columns that were examined at were: Kromasol C18, Develosil C18, Sunsil C18, Aligent C18 and Supelco C18. All examined stationary columns have identical dimensions (5 μm; 4.6 × 250 mm). The solvent combinations that were examined at were: 0.1% H3PO4-methanol; 0.1M NaH2PO4methanol; and 0.1M Na2SO4-acetonitrile. The temperature (25˚C) at the stationary column, column flow stream (1 ml/min), and injection analysis volume (10 µl) were all held steady throughout the trial experimentations. Finally, the settings explained in section "Instrumentation and analysing conditions of TELM and AZEL combination" were opted to analyse the TELM and AZEL combination.
No peaks were spotted nearby to the RT of TELM and AZEL in diluent (55% volume 0.1M Na2SO4 with pH 3.6 and 45% volume acetonitrile), approximately the same RT of (55% volume 0.1M Na2SO4 with pH 3.6 and 45% volume acetonitrile) in standard TELM and AZEL solution and formulation solution. Hence evidenced high selectivity of TELM and AZEL analysing method [20].
The TELM and AZEL's concentration was evidently linear for the proposed HPLC-based stability demonstrating approach in the possibility of 20-60 µg/ml and 4-12 µg/ml, with strong linearity as correlation coefficient was 0.9996 and 0.9980, respectively [21].
The low quantification and detection limits concentration values were considered reasonable, ample and sensitive for the TELM and AZEL analysing method [22].
The RSD assessments for the TELM and AZEL peak response were within 2%, indicating that the HPLC-based stability demonstrating process was precise and repeatable for determining TELM and AZEL [23]. The recovery assessments for the TELM and AZEL assay within 100±2% recommended that HPLC based stability demonstrating approach was accurate and reliable for determining the TELM and AZEL [23].
The misplays in expressions of RSD for TELM and AZEL peak response after making some modest modifications in acetonitrile ratio (1.3% for TELM and 1.8% for AZEL), column rate of flow (1.8% for TELM and 1.7% for AZEL) pH (0.5% for TELM and 0.6% for AZEL), column temperature (1.7% for TELM and 1.9% for AZEL) and wavelength (1.4% for TELM and 1.7% for AZEL) within 2%, indicating that the HPLC based stability demonstrating process was robust for determining the TELM and AZEL [25].
The choice of formulation production, storage, shipping, shelf life, packaging, and chemical stabilization of TELM and AZEL is directly influenced by stability testing [13][14][15][16]26]. The TELM and AZEL were realised more stable under oxidation degradation testing conditions employed. The TELM and AZEL was realised more sensitive under heat degradation testing situations employed. The retention times of TELM, AZEL and degradation products got during acid degradation testing, oxidation degradation testing, alkaline degradation testing, photodegradation testing, and heat degradation testing conditions are completely different. Hence evidenced high stability demonstrating feature and specificity of TELM and AZEL analysing method.

CONCLUSION
A stability demonstrating methodology for analysing the TELM and AZEL combination in the tablets and bulk was elucidated in this report. This method found fit, based on the values obtained during validation experiments, for the quality regulatory analysis of TELM and AZEL combination in the quality declaration laboratories deprived of interference from pharmaceutical excipients of TELMA-AZ tablets and degradation products got in acid degradation testing, oxidation degradation testing, alkaline degradation testing, photodegradation testing, and heat degradation testing conditions employed.

AUTHORS CONTRIBUTIONS
All the authors have contributed equally.

CONFLICTS OF INTERESTS
Declared none