Quantification of Telmisartan and Azelnidipine Combination in Using Liquid Chromatography: Stability studies

A stability-indicating RP-HPLC method for the development of Telmisartan (TTN) and Azelnidipine (ADN) is analyzed in tablet dosage form. The quantification of TTN and ADN combination is done by Supel cosil C18 column (250 mm, 4.6 mm, & 5 µm). Isocratic mobile phase had mobile phase consists of 0.10M Na 2 SO 4 (pH 3.6) and acetonitrile (pH 3.6) as 55:45v/v. For this analysis flow rate is measured as 1.00 mL/minute. Wavelength is identified as 258nm to examine TTN and ADN. Stability for both these drugs under distinctive environments were performed. Injected volume is 10 µL. Run time is 8 minute. Retention time is 2.8 and 3.7 respectively. The responses were linear in the concentrate range as 37.4-110.3 for TTN and 2.24-10.51 µg/mL for ADN respectively. Percent comparative standard deviance to precision is 0.193% for TTN, 0.195% for ADN. Percent assay to accuracy for both these drugs are 98.76% and 99.04% respectively. LOD values for TTN and ADN were 0.020 µg/mL and 0.065 µg/mL and LOQ values for TTN and ADN were 0.009 µg/mL and 0.031 µg/mL. Robustness studies revealed that this method is robust by percent comparative standard deviance. This stability-indicating RP-HPLC procedure to both TTN, ADN analysis is more simple, highly sensitive, more precise, highly specific and robust, making it appropriate to the assessment of TTN and ADN in formulation.


INTROdUCTION
Telmisar tan is chemically approved and the molecular formula is C 33 H 30 N 4 O 2 and the molecular weight is 514.62.TTN is acting as receptor inhibitor for angiotensin II 1,2 .It consists of very broad area of dissemination which is matured to its very strong property as lipophilicity along with a final eradication halflife as one day 1,2 .This also maintains very good blood pressure after one missing dosage.TTN by hydrochlorothiazide mixture provides in larger minimization in final 720 seconds average systolic and diastolic blood pressure 3,4 .The dosage of this drug is usually taken with the help of mouth. 5his TTN is available in different versions includes telmisartan/hydrochlorothiazide, telmisartan/ cilnidipine 6 and telmisartan/amlodipine. 5 Molecular formulae for Azelnidipine(ADN) is C 33 H 34 N 4 O 6 and molar mass is 582.657g•mol −1 .This drug is a decrease in blood pressure by comparable potency with various dihydropyridines, includes amlodipine, without pulse rate increasing 7 .Azelnidipine (ADN) is a dihydropyridine-kind calcium antagonist that may be newly developed to treat hypertension 8,9 .S. Yuvasri 10 et al., proposed total two methods.Wavelengths are 324nm for TEL and 220nm to AZEL are identified.In the second method various derivatives are noted as 220nm to TEL as well as 244nm to AZEL.Linearity is 16-80 µg/mL to TEL, 3.2-16 µg/mL for AZEL.Parikh MB 11 et al., separated by using ODS C18 (250*4.6mm)and Buffer as 0.05M KH 2 PO 4 buffer with pH-4.0 methanol as 60v/v, 40v/v as mobile phase, rate of flow as 1.00 mL/minute.215nm is wavelength.RT values are 3.440 min and 5.693 minutes.Linearity is 20-60 µg/mL and 40-120 µg/mL.M. S. Kalshetti 12 et al., developed method to Metoprolol succinate, Telmisartan, as well as Clinidipine using Phenomenex Luna C18 (150mm×4.6mm,5 µm) as stationary phase.Acetonitrile, methanol and phosphate buffer are used in the ratio of 45:30:25v/v/v maintained at pH range as 7.5.Flow rate is maintained at 1.00 mL/minute.Wave length is 229nm.Total drugs were eluted at 2.0 min, 2.8 min and 6.8 minute.Linearity range is 10-80 µg/mL, 6.25-50 µg/mL and 2.5-20.00µg/mL by coefficients of regression 0.997, 0.995 and 0.999 to telmisartan, metoprolol succinate as well as clinidipine.Jayvadan K Patel 13 et al., used Hypersil GOLD C18 (150mm×4.6mmwith internal diameter, as well as size of the particle as 5 µm).Mobile phase is methanol 40v/v, acetonitrile 40v/v, and water 20v/v.The rate of flow is 0.50 mL/minute.260nm is the wave length.RT values are 8.56, 3.04 minute.Curves of calibration are 2-48 µg/mL to AZL and 2.5-60 µg/mL to OLM by correlation

MATERIAL ANd METHOdS
Telmisar tan and Azelnidipine were procured from local market, Hyderabad, India, as a gift sample.0.10M Na 2 SO 4 , acetonitrile of HPLC Grade, 1N KOH (AR Grade), Buffer (AR Grade) were purchased from E. Merck (India) Ltd.Mili-Q water was used throughout the experiment.For analysis, drug product substance named Uniaz T40 solution comprising 40 g/mL TTN and 8 g/mL ADN was produced.

ExpERIMENTAL
A "Waters" HPLC system (model-2695) fitted out with a PDA (model-2998) detector was exploited for analysing TTN & ADN in Tablet of brand Uniaz T40.TTN and ADN were separated chromatographically with the help of a Supelcosil C18 column (250mm, 4.6mm, & 5 µm).At stream rate as 1.00 mL, mobile phase was constituted of 0.10M Na 2 SO 4 having a pH of 3.6 and acetonitrile (55:45, vol/vol ratio).The volume of TTN & ADN solution injection was 10 µL.The temperatures of the column and also the autosampler tray remained maintained at 27°C all through the experiments.Weighed accurately 14.2 g of Na 2 SO 4 taken into a beaker and then coefficients value more than to 0.990.Other researchers 14,15 are developed different methods for the determination of various drugs.By using literature reviews authors are carried out this method for the development of telmisartan and azelnidipine.added 1000 mL Milli-Q water along with dissolved it then altered pH 3.6 by using 1 N KOH solution and then filtered buffer solution over 0.22 µm filter paper.Mixed volumes of 550 mL Buffer solution and 450 mL acetonitrile into a 1 litre bottle and then degassed the mobile phase by using sonication.

Method development
For 10ppm solution of TTN as well as ADN by using UV spectrophotometer the spectrum in Acetonitrile was recorded separately.Peak of maximum absorbance wavelength was measured at 258nm absorbance indicated the spectra of TTN as well as ADN.By using Supelcosil C18 column column required separation along with peak shapes were measured.In this method mobile phase was constituted of 0.1 M Na 2 SO 4 having a pH of 3.6 as 55v/v and acetonitrile as 45v/v.Finally, noted from experiment is 1.00 mL/min flow rate is most suitable in order to elute analyte.The obtained chromatograms are denoted in Figure 2.

System suitability
This parameter is inspected with working solution of TTN as 20 µg/mL as well as ADN as 10 µg/mL.Mean, SD along with %RSD to resolution, area to peak, period of retention, symmetry for peak along with theoretical plate number are measured and the results were denoted in the Table 1 & 2 to both TTN as well as ADN peaks conferring to ICH indorsed criteria.The obtained chromatograms are denoted in Figure 3.

Specificity
Examined and determined parameter to this proposed process after injecting Diluent as blank, construction of placebo, solution for system suitability, standard solution-diluted, sample construction as well as disparate Impurity's includes A, B, C, D, E as well as construction of spiked sample into the system of chromatography latter retention times are reported.From the values obtained finally it is noticed that there is no possibility for the presence of other interference by Diluent-blank, construction of placebo, solution of system suitability, standard solution-diluted, sample construction and divergent Impurity's like A, B, C, D, E at Rt of TTN and ADN peak together with each other.The final values were noted in the Table 3.  4.
Among diverse C18 columns (Kromasil, Develosil, Sunsil and Supelco) investigated, better resolution of TTN and ADN peaks, as well as peak symmetry for TTN and ADN were obtained with Supelco.TTN and ADN have the maximum sensitivity at 258nm, with the least amount of observed noise.TTN and ADN peaks were not eluted adequately with 0.1% phosphoric acid and 0.1M NaH 2 PO 4 and provided an unsatisfactory baseline.As a consequence, 0.1M Na 2 SO 4 having pH 3.6 was chosen since it produced a superior outcome.The resolution of TTN and ADN peaks, as well as peak symmetry for TTN and ADN, were markedly enhanced while acetonitrile was added in a 45% volume ratio to mobile phase.Chromatogram of TTN and ADN with optimized conditions is made known by the Figure 5.

Linearity
Carried out linearity with both TTN as well as ADN to 300% of impurity specification limit.Parameter like precision is measured at maximum level.Correlation coefficient and R square value is minimum to 0.995.The %intercept is below to 5.0 of response at 100% specification area.Precision for maximum levels of %RSD is NMT 5.0.Calibration curve is acquired to both TTN as well as ADN by injection as 10 µL volume of calibration BLS range as 10.00 µg/mL-30.00µg/mL along with MTL range as 5 µg/mL-15.00µg/mL solutions.TTN as well as ADN area below their peaks was marked selected against corresponding TTN as well as ADN strengths are indicated in the Fig. 81

Limit of detection & quantitation
With the help of intercept, slope along with residual regular deviation computed LOD and LOQ.Values to LOD as well as LOQ for both TTN along with ADN are profient which is dependent over area response standard deviation as well as calibration graph slope.Values to LOD for both TTN as well as ADNare 0.0180 µg/mL and 0.0240 µg/mL, respectively.The values to LOQ for both TTN as well as ADNwere 0.0590 µg/mL and 0.0810 µg/mL, respectively.The results are tabulated in the Table 4.The evaluates of the LOD and LOQ are 0.0200 µg/mL and 0.0650 µg/mL for TTN.The evaluates of the LOD and LOQ are 0.0090 µg/mL and 0.0310 µg/mL for ADN.These evaluates of TTN & ADN confirming the sensitivity.The results are shown in the Table 4.

Accuracy
The accuracy for TTN & ADN from developed chromatograms were ascertained, and measured as a function of %TTN & ADN assay.These evaluates of %TTN & ADN assay confirming the accuracy.The obtained graphs are represented in the Figure 9.

Recovery
Recovery investigations were taken to ensure accuracy.A measured quantity of TTN & ADN was spiked into a pre-quantified Uniaz T40 formulation (40 µg/mL-TTN and 8 µg/mL-ADN) at different potencies (50%, 100%, and 150%) level.The Uniaz T40 formulations spiked were diluted and chromatograms were generated under appropriate TTN & ADN assay chromatographic conditions.The peak areas for TTN & ADN were observed, and using a regression models or calibration curves, the amount of TTN & ADN were calculated.These evaluates of %recoveries of TTN & ADN assay confirming the selectivity.The recovery results are denoted in Table 6.

Robustness
Total known impurities may be separated from each other latter to both TTN as well as ADN peak in sample that is spiked by impurities.Factors endorsed to evaluate robustness are: abnor mality in wavelength as +2nm and −2nm, flow rate as +0.10 mL/min and −0.10 mL/min, methanol proportion as +5% volume and -5% volume, pH as +0.10 unit and -0.10 unit and column temperature as +2 0 C and −2 0 C. Robustness is authorized by working TTN as 20.00 µg/mL along with ADN as 10.00 µg/mL solution.Outcome of altered factors over analysis  of TTN as well as ADN is evaluated in relations of Mean, SD and %RSD for TTN as well as ADN peak areas captured along with the results shown in the Table 7.

RESULTS ANd dISCUSSION
Here in investigation, we designed an HPLC approach for detecting and analysing TTN and ADN in bulk & tablet doses.This HPLC approach was sensitive, having a quantification level of slightly below 0.1 µg/mL.This HPLC TTN and ADN assay technique can be incorporated in a few of the very precise, accurate, selective & sensitive procedure listed for TTN and ADN analysis, according to observed findings of validation criteria.These characteristics recommended that the presented technique be utilized in analytical quality assurance (QA) procedures that are frequently performed by regulatory bodies and QA laboratories, without the influence of some frequently used dosage additives.Herein investigation, we designed an HPLC approach for detecting and analysing Telmisartan (TTN) and Azelnidipine (ADN) in bulk & tablet doses.TTN and ADN were separated chromatographically using a Supelcosil C18 column and mobile phase was constituted of 0.10M Na 2 SO 4 having pH 3.6 and acetonitrile (55:45, vol/vol ratio).
Linear relationships for these drugs obtained around concentration ranges 20-60 µg/mL and 4-12 µg/mL.The calibration factors demonstrated are 29431 and 181619 slope values and -24712 and 4475.2 intercept values for TTN and ADN, respectively.Evaluated %RSD for TTN as 0.110% & ADN as 0.193% confirmed the precision.The evaluates of %TTN as 98.78% and for ADN as 99.13% assay confirmed the accuracy.Forced degradation assessments were carried out on TTN & ADN as per ICH directives.The stability indicating property was revealed by a well-resolved TTN, ADN peaks N as well as degradation products at various elution periods.The precision examination includes estimating TTN as 40 µg/mL & AND as 8 µg/mL drug solutions six times on relatively similar day.The Uniaz T40 formulations spiked were diluted and chromatograms were generated under appropriate TTN & ADN assay chromatographic conditions.The peak areas for TTN & ADN were observed, and using a regression models or calibration curves, the amount of TTN & ADN were calculated.
Factors endorsed to evaluate robustness are: abnormality in wavelength as +2nm and −2nm, flow rate as +0.10 mL/min and −0.10 mL/ min, methanol proportion as +5% volume and -5% volume, pH as +0.10 unit and -0.10 unit and column temperature as +2 0 C and −2 0 C. Robustness is authorized by working TTN as 20.00 µg/mL along with ADN as 10.00 µg/mL solution.Outcome of altered factors over analysis of TTN as well as ADN is evaluated in relations of Mean, SD and %RSD for TTN as well as ADN peak areas captured.The presented technique be utilized in analytical quality assurance procedures that are frequently performed by regulatory bodies and quality assurance laboratories.

CONCLUSION
According to ICH guidelines, this HPLC method for the associated compounds in drug product of TTN & ADN is verified.The proposed procedure has been determined to be specific.The approach is also shown by stressful circumstances.

Fig. 2 .Fig. 3 .
Fig. 2. Different Flow rate chromatograms & 82.Linear relationships for TTN as well as ADN were obtained around concentration ranges 20-60 µg/mL (TTN) and 4-12 µg/mL (ADN) by reliable regression as 0.9998 for TTN and 0.9999 for ADN coefficients.The calibration factors demonstrated are 29431 and 181619 slope values and -24712 and 4475.2 intercept values for TTN and ADN, respectively.The linearity curves are represented in Fig. 6 & 7 and chromatograms are represented in Figure 8.