Rapid RP-HPLC Method For the Dual Drug Analysis of Azelnidipine and Chlorthalidone with Stability Insights

Hypertension is one of the significant reasons for the increased death rate in the previous 10 years and is expanding dramatically because of the ongoing undesirable lifestyle [1]. It is a major risk factor for stroke, heart failure, kidney disease, and premature death [2]. Hypertension is a common disease that is simply defined as persistently elevated arterial blood pressure [3]. Blood pressure is created by the force of blood pushing against the walls of blood vessels (arteries) as it is pumped by the heart [4]. To reduce the risk of cardiovascular disease, stringent blood pressure management is acceptable by combining a few different antihypertensive medications [5]. To manage hypertension, Calcium Channel Blockers have been frequently co-administered and also reduce cardiac contractions by blocking calcium channels in the myocardial and vascular smooth muscles [6].  Chlorthalidone, as 2-chloro-5-(l-hydroxy-3-oxo-2,3-dihydro-1H-isoindol-1-yl) benzene-1-sulfonamide, is an oral thiazide diuretic that is reliably effective in most patients with high blood pressure, and is considered as preferred initial treatment directed alone and in combination with antihypertensive specialists [7]. Chlorthalidone inhibits sodium reabsorption at the level of the distal convoluted tubule and thus chloride via inhibition of the Na-Cl symporter [8].

Fig. No.1 Chemical structure of Chlorthalidone

Synthetically, Azelnidipine is 3-[1-(Benzyldrylazetidin-3-yl] 5-isopropyl-2-amino-6-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate ^[9]. A calcium channel blocker classified as a dihydropyridine is Azelnidipine ^[10]. In vascular walls, Azelnidipine prevents transmembrane Ca²⁺ influx through smooth muscle voltage-dependent channels and is a vasodilator that lowers blood pressure gradually in people with hypertension ^[11]. It has been demonstrated that calcium channel blockers slow atherogenesis in animal models and stop the onset of early lesions in human coronary arteries ^[12].

Fig. No.2 Chemical structure of Azelnidipine

MATERIALS AND METHODS:

Chemicals and reagents:

Analytically pure standards of Chlorthalidone and Azelnidipine were obtained from Aavyan Labs, Hyderabad. Azelnidipine and Chlorthalidone in combination (labelled claim amount of 8mg Azelnidipine and 12.5mg Chlorthalidone per tablet) was procured from Torrent Pharmaceuticals, Uttarakhand, India. HPLC-grade water, methanol, acetonitrile, and acetone were purchased from Research Lab Fine Chemicals, Mumbai, and distilled water from the local market.

Equipment’s:

Ultra-Fast Liquid Chromatography -Shimadzu with LC Solution as software, FTIR-Bruker ATR Alpha interferometer attached to OPUS software, UV Spectrophotometer- Shimadzu UV-1700, Pharmaspec Japan UV-Probe as software, electronic balance Tandem TJ Series, vacuum filter, Equitron Ultrasonic Cleaner. pH meter -Systroniks MKV1, Hot air oven- Kemi medicals, KOA 3.

Chromatographic conditions:

Shimadzu UFLC model with Phenomenex C₁₈ (250mm×4.6mm;5µm) column was used for the separation of Azelnidipine and Chlorthalidone with a mobile phase of Methanol: Water (98:2%v/v), and the flow rate was set at 1.1mL/min with an injection volume of 20µL, at a λmax of 236nm and run time of 4 min. HPLC-grade methanol was used as the diluent.

Preparation of a standard solution of Azelnidipine and Chlorthalidone:

The standard stock solution was prepared by accurately weighing and transferring 10mg of Azelnidipine and 10mg of Chlorthalidone pure drugs into two separate 10 mL standard flasks. About 4mL of diluent was added, and the solution was ultrasonicated for 10 min to dissolve the drugs completely. And the final volume was made up of HPLC-grade methanol (Azelnidipine 1000µg/mL, Chlorthalidone 1000µg/mL). Accurately, .2mL of standard stock solution of each was transferred into a 10mL standard flask, and the volume was made up with the diluent to prepare a working standard solution of 20µg/mL.

Preparation of a sample solution of Azelnidipine and Chlorthalidone:

About 20 tablets of a fixed-dose combination of Azelnidipine and Chlorthalidone 8mg and 12.5 mg, respectively, were weighed and powdered. Tablet powder equivalent to 8mg Azelnidipine and 12.5 mg Chlorthalidone was weighed and transferred into a 10mL standard flask and dissolved completely by sonicating for 15 minutes using diluent and filtered the stock solution. About .08mL stock solution transferred to a 10mL standard flask, and the final volume made up with HPLC-grade Methanol. Working sample solution: 8µg/mL of Azelnidipine and 12.5µg/mL of Chlorthalidone were obtained.

Validation ^[13]_:

The developed RP-HPLC method was validated as per ICH guidelines. The parameters validated are Specificity, Forced degradation studies, Accuracy, Precision (Intraday precision, Inter-day precision), Linearity, Limit of Detection (LOD), Limit of quantitation (LOQ), Robustness, and System suitability parameters.

Specificity:

The specificity of the developed RP-HPLC method was established by injecting 20 μL each of the blank, working standard, and sample solutions.

Forced degradation studies ^[14]:

Forced degradation studies of the drug in the presence of an acid, alkali, H2O2, temperature, and HPLC-grade water were performed to establish the stability of the developed method.

Acid degradation:

1 mL of standard stock solution of Azelnidipine and Chlorthalidone (100µg/mL) was treated with 1 mL of.1M HCl and refluxed for 30 minutes to assess degradation under acidic conditions. The solution was neutralized with 0.1N NaOH. With the diluent, the resulting solution was diluted to 10 mL.

Alkali degradation:

Degradation was studied under alkaline conditions by refluxing 1 mL of standard stock solution of Azelnidipine and Chlorthalidone (100µg/mL) with 1 mL of.1M NaOH for 30 min, neutralized with 0.1N HCl the diluent was used to dilute the stressed solution to 10 mL.

Oxidative degradation:

About 1 mL of Azelnidipine and Chlorthalidone standard stock solution (100µg/mL) was treated to oxidative degradation by refluxing with 6% v/v H₂O₂ in a 10-mL volumetric flask for 30 min and then made up with the diluent.

Thermal degradation:

By heating the normal stock solution to 40, 60, and 80 degrees Celsius in the oven, thermal stability was assessed. The diluent was used to dilute 10 mL from around 1 mL of the stressed solution.

Neutral degradation:

 In a 10-mL volumetric flask, 1 mL of standard stock solution was refluxed with 1 mL of HPLC-grade water to perform neutral degradation. The diluent was used to make up for the volume. Approximately 20µL of each of the solutions subjected to different stress conditions was injected individually into the column, and the chromatograms were recorded to assess the drug's stability.

% Degradation=( Peak area of untreated stock solution-peak area of treated stock solution)/(peak area of untreated stock solution)×100

Accuracy:

Azelnidipine and Chlorthalidone were injected 20µL into the column three times at concentrations of 80, 100, and 120% to determine accuracy. Using the peak area at each level, the average percent recovery of the three levels was calculated.

Precision:

By injecting six samples of a working standard solution of azelnidipine and chlorthalidone into the column on the same day for intraday precision and on two consecutive days for interday precision, the precision of the improved technique was assessed. % RSD was computed.

Linearity:

The linearity of the developed technique was tested by injecting Azelnidipine at concentrations ranging from 20 to 120 µg/mL and Chlorthalidone at concentrations ranging from 20 to 120 µg/mL. The calibration curve was created by graphing the peak area on the y-axis vs concentration (µg/mL) on the x-axis.

The correlation coefficient of the calibration curve was calculated.

Table 1: Linearity trials of Azelnidipine

Table 2: Linearity trials of Chlorthalidone

LOD and LOQ ^[15]:

LOD and LOQ were calculated using the formula based on the standard deviation of the y-intercept of regression lines and the slope of the calibration curve.

LOD=3.3 × σ/ S

LOQ=10 × σ/ S

where σ is the standard deviation, S is the slope of the curve

Robustness:

Robustness of the developed RP-HPLC method was evaluated by making minor changes in the flow rate (±.1mL/min), change in mobile phase ratio, and wavelength (±5nm). The parameters evaluated were % RSD of peak areas, theoretical plates, tailing factor, and resolution.

Ruggedness:

A sample solution containing 100 µg/mL was injected six times to test the ruggedness. Two different analysts were used for the analysis, so the system under study underwent two rounds of analysis. The test's outcomes were found to be within limits, and the %RSD value was less than 2.

System suitability testing:

A working standard solution was injected into the column to test the system suitability parameters like % RSD of peak areas, theoretical plates, tailing factor, and resolution under optimal chromatographic conditions.

Assay ^[16]:

The sample solution was injected six times into the column, and the % assay was calculated by using the formula:

%Assay=[(sample area)/(standard area)]×[(dilution of standard)/(dilution of sample)]×(p/100)×(avg. wt)/LC×100

• Avg. wt. is the average weight of tablets
• P is the percentage purity of the working standard
• LC is the label claim of the drugs

RESULTS:

Based on the results of the solubility tests, HPLC-grade Methanol was chosen as the diluent. In methanol, both Azelnidipine and Chlorthalidone are easily soluble.

Method optimization:

The trial-and-error technique optimized the process to get a chromatogram with excellent resolution, an appropriate number of theoretical plates, and a tailing factor. Preliminary trial runs were undertaken to optimize the technique by varying the mobile phase ratio and run time. The PDA detector has the benefit of capturing chromatograms in the whole UV spectrum in a single run. The optimal λmax was discovered to be 236nm. UV spectrum of Azelnidipine is shown in Fig.3, Chlorthalidone in Fig.4, and the overlay spectrum in Fig.5. Solvents like methanol and water in various ratios were tested on a C18 column during the trial runs to determine the best separation of the chosen combination of drugs. This improved technique used methanol: water (98:2% v/v) as the mobile phase because the resolution and peak shape of Azelnidipine and Chlorthalidone were satisfactory with optimal system suitability parameters. The flow rate was chosen based on the peak resolution and the lowest consumption of the mobile phase, which was 1.1 mL/min. Chlorthalidone and Azelnidipine were eluted at 2.246 min and 2.819 min. The chromatograms showed no extra peaks, suggesting that there was no excipient interaction with the medications at the retention length of time, and the method was created specifically for the simultaneous estimation of the pharmaceuticals in tablets. Studies on forced degradations indicated that the purity threshold for both drugs was higher than the purity angle, indicating that there was no co-elution of degradants with drugs. The analyte peaks and the drugs were both pure. For both drugs, a percent degradation of less than 10 indicates that the established analytical method was accurate and reliable (Table 4,5).

Fig. No.3 UV spectrum of Azelnidipine Fig. No.4 UV spectrum of Chlorthalidone

Fig. No.5 Overlay spectrum of Azelnidipine and Chlorthalidone   Fig. No.6 Optimized chromatogram of Chlorthalidone and Azelnidipine

Table 5: Forced degradation of Chlorthalidone

Table 6: Accuracy (% recovery of Azelnidipine and Chlorthalidone)

Table 7: Linearity of Azelnidipine and Chlorthalidone

Fig. No.7 Calibration curve of Azelnidipine and Chlorthalidone

DISCUSSION:

Stability-indicating assay method development examines the effect of stressors on a drug, which aids in understanding the drug's stability throughout storage and analysis. There aren’t many techniques available for estimating Azelnidipine and Chlorthalidone by RP-HPLC separately. Azelnidipine and chlorthalidone were eluted at 2.819 and 2.246 min, respectively, with a run duration of 4 min. The current approach was created with methanol and water as the mobile phase. The established technique was shown to be cost-effective since it required minimal sample preparation and the use of readily available solvents and materials.

Table 9: Results of the robustness of Azelnidipine