Development and Validation of RP-HPLC Method for Simultaneous Determination of Saroglitazar and DPP4 Inhibitors

INTRODUCTION

Form 2 diabetes mellitus (T2DM) is more than 70% the main cause of cardiovascular disease (CVD) and CVD related morbidity. Dyslipidemia attributable to T2DM is one of CVD's main comorbidities [1,2]. Currently, several anti-diabetic medications are used to avoid symptoms in diabetics. Single treatment counselling, though is not adequate to inhibit metabolic syndrome triggered by diabetes. Thus an adequate medication combination treatment must be found that has substantial success in reducing diabetic complications. Peroxisome-activated receptor α/μ(PPARα/μ) dual agonist, saroglitazar is used in diabetic dyslipidemia & hypertriglyceridemia[3,4]. [3,3]. Saroglitazar is biologically a benzenepropanoic acid, alpha-ethoxy-4-[2-[2-methyl-5-[4-(methyl)phenyl]‐1H‐pyrolean-1-yl]ethoxy]– magnesium salt, shown in figure 1,[4] Saroglitazar, due to its advantages in significant triglyceride and low level lipoprotein [4,6], is the first Glitazar-class medicament approved for the treatment of diabetic dyslipidemia. Clinical studies have documented a positive impact of the T2DM patients' saroglitazaron lipid and glycemic parameters without any side effects such as infibrates and pioglitazone medications. In addition, there was no chance of associated hypoglycaemia [7,8,9]. In Phase III clinical trials, the Saroglitazar Prospective Randomized Effectiveness and Protection [PRESS] V study and the PRESS VI analysis, the safety, efficacy and tolerability of saroglitazzar action have been established via the unique receptors [10,11] of PPARα. Saroglitazar's dual activity allows it a promising molecule for controlling the cardiovascular danger of T2DM[12,13]. DPP-4 inhibitors offer a novel clinical strategy to managing type 2 diabetes, which acts by inducing glucose-dependent insulin release and reducing glucagon levels. In specific, glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) are obstructed by inhibition of the glycemic controls. (14) Chemically, linagliptin is 8-[(3R)-3-aminopiperidine-1-yl]-7-(but-2-yn-1-yl)-3-methyl-1-[(4-methylquinazoline-2-yl) ethyl]-3,7-dihydro-1H-purine-2,6-dione] (Figure 1) and is dipeptidylpeptidase-4 inhi-a. The special structure, distinguished by five consecutives Rings (Figure 1) and peptidomimetic [17], is Teneligliptin, {(2S,4S)-4-[4-(3-methyl-1-phenyl-1 H-pyrazol-5-yl)piperazine-1-yl]Pyrrolidine-2-yl}(1,3-thiazolidine-3-yl). High-performance liquid chromatography (HPLC), for the calculation of analyte in the difficult

to establish an analytical method for the removal of matrix interferences. No HPLC method for the simultaneous determination of saroglitazar and DPP4 inhibitors is currently known. In this research, a modern, accurate, fast, robust, reproducible and economic method for the simultaneous determination of Saroglitazar and DPP4 inhibitors using RP-HPLC was established and validated.

As a gift sample, saroglitazar (Cadila Healthcare Ltd., Vadodara, Gujarat, Inde), Linagliptin (Glenmark Generics Ltd., Bharuch, Gujarat, India) and Teneliptin (Glenmark Generics Ltd., Bharuch, Gujarat, India) were obtained.

The method was developed using the auto sampler, online degaasser and 2998 photodiode array (PDA) detector device of Waters ez695 partnership with HPLC.

Phenomenex Luna C-18 (250 to 4.6 mm, 5 μm) with a gradient flow pump was used for chromatographic separation. The wavelength was calculated by PDAscanning at 200 nm to 400 nm and for further study 243 nm was chosen for anoptimum reaction. Originally, the process formulation was carried out by the combination of water: acetonitrile with various ratios, but none of the ratios were able to provide a right picture resolution. The mobile step pH of the RP-HPLC is avital variable in retention control as the sample comprises ionic or ionizable compounds. A range of research was conducted utilising phosphate buffers with altered pH along with various percentages of acetonitrile and in the pH 7.0 of the mobile phase A and acetonitrile phase B greater isolation and peak symmetry than others. The mobile phase proportion was optimised with a variety of phosphate buffers with pH 7.0 and acetonitrile. Finally, the phosphate buffer (pH 7.0) and acetonitrile (different ratio) were found to be useful for evaluating inhibitors of saroglitazar and DPP4 (Table 1). The amount of injection was 10 μL. The phase period was 12 minutes and the ambient temperature was held throughout the study.

A stock solution of saroglitazar, linagliptin and teneligliptin was prepared by specific weighing of 1 mg API of saroglitazar, linagliptin and teneligliptin in the dark before study. The stock solution was used for the calibration analysis for progressive dilutions.

RESULTS:

The predictability of an analysis methodology is its capacity to yield effects that are equivalent to the sample concentration of an analyte in a specified range, directly or by a well-defined statistical conversion. By evaluating the various concentrations of a regular saroglitazar solution, linagliptin and teneligliptin linearity were assessed. The solution of the triplicate and curved API injected by saroglitazar, linagliptin and teneligliptin was obtained by contrasting the image with the concentration of a substance. The spectrum of linearity was 0.1-0.5 μg/mL. A linear method for saroglitazar, linagliptin and teneligliptin with R2>0.999 was discovered. The system developed was reliable with R2>0.99 and relative standard deviation (RSD)<2 percent.

The precision of the calculation was evaluate by measuring 3 replicates of each low quality control (LQC) concentration at different periods on the same day (intra-day) and on different days (inter-day) and its standard deviation(SD) and RSD percent at different intervals and the findings were presented in Table 2.

The consistency of a research system represents the closeness to the true meaning of the test outcomes produced by a process. Accuracy may be calculated through applying the analytical technique to an established pureness assay (for the medication substance) or by remediation tests with a defined normal increase in placebo (for the pharmaceutical). The accuracy has been determined in the current research by recuperation studies (50%, 100% and 150%) by taking various quantities. The estimated recuperation percentage values are displayed in Table 3.

The detection maximum is the volume pumped that exceeds a peak height of at least two to three times the Baselinenoise frequency (Signal/Noise = 2 – 3). LOD is the lowest quantity of a sample that can be detected and not quantified considerably. The LOQ determines the lower study in a study that can be quantitatively calculated with sufficient precision and accuracy (signal/noise = 10-20). In this analysis, LOD and LOQ were calculated by steadily injecting a low concentration of the regular analyte solution. The LOD value for saroglitazar, tenneligliptin and linagliptin was observed to be 0.037μg/mL, 0.014μg/mL and 0.096μg/mL. A combined LOQ value of 0.188 μg/mL, 0.074 μg/mL and 0.482 μg/mL was identified (Table 4).

Except for talc‘s, magnesium stearate, povidone, microcrystalline cellulose (MCC), colloidal siliqua and croscarmellose sodium, excipients widely used in tablet formulation were mixed. The regular solution with a known saroglitazar concentration was spiked into the mixture and analysed using the method established. The spiked solution chromatogram was equivalent to the normal solution chromatogram for measurement. Following reference, the analytical approach developed was found to be specific, since no intervention was detected during the preservation of all three analytes.

The robustness is an analytical tool which tests the method's ability to remain unchanged by a slight yet deliberate variance, i.e. pH, flow rate, the column temperature and injection capacity. It also shows the efficiency of the operation by regular use. The robustness of the system developed was seen in Table 5 at the variant flow rate and mobile step composition.

Ruggedness is a test of effects replicability in multiple environments, such as climate, labs, analysts, equipment, operators and materials. The ruggedness of the approach built was tested by analysing the outcomes of intraday and inter-day analyses carried out by two separate analysts. The RSD percentage was less than 2 percent suggested that the method built was robust enough.

Table 6 was determined on the suitability of the device and chromatographic parameters, for example, astail factor, asymmetry factor and number of theoretical plates. conditions for preclinical pharmacokinetic tests. The findings of the present research indicate that the approach being implemented is simple, fast, economical, sensitive, reliable and precise. It can also effectively be used for routine review in quality management. The findings indicated that the procedure proposed was sufficient for simultaneous determination of inhibitors saroglitazar and DPP4. In conjunction with acetonitrile organic solvent, various mobile phases were used. Among them is the composition of mobile phase mixture, which resulted in a phosphate buffer (pH 7.0): a 25:75 (percent v/v) ratio, which provided a sufficient resolution of saroglitazar peak, linagliptin, and teneligliptin. The flow rate in the established system of 1 mL/min and 12 minutes suggests that the method developed is economically approved. The procedure was tested in compliance with the ICH guideline[18] in order to make it technically appropriate. The calibration curve was illustrated against the concentration of the standard solution by drawing standard peak regions. Low percent intra-day and inter-day RSD values have shown that the approach established is reliable. Recovery experiments were carried out by analysing analytical findings for the recovered samples at three stages (50%, 100% and 150%) to maintain accuracy and reproducibility. The findings of the precision evaluation were also in line with the ICH approval criterion. Repeatability of the highest zones, tail factor and number of theoretical plates (N) examined in the Device Test (SST) suggested that the procedure for simultaneous saroglitazar, linagliptine, and teneligliptin determination was sufficient.

For the identification of saroglitazar, linagliptine and teneligliptin, a modern, accurate and efficient RP-HPLC method was created. The key advantage of the approach developed is cost-effectiveness, quick examination, sufficient consistency and precision. The reproducibility of this approach is distinguished by low RSD values intra-day and inter-day examination and excellent recoveries. High quality data and critically tested methods represent the highly responsive, precise and rigorous methods developed. Considering all the parameters of the validated process, it seems quite useful in raw material and dose research at different stage in industry for saroglitazar, linagliptin and teneligliptin.

The authors are rather appreciative of the provision of DPP4 API inhibitors by Cadila Healthcare

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Departments of Pharmacology, L. M. College of Pharmacy, Ahmedabad, Gujarat, India dranitalmcp@gmail.com