Simultaneous Determination of Linagliptin and Metformin by Reverse Phase-High Performance Liquid Chromatography Method: An Application in Quantitative Analysis of Pharmaceutical Dosage Forms

Abstract

Linagliptin and Metformin are approved drugs for the treatment of type 2 diabetes mellitus. They are both used in the same dosage forms. We report here that a High-Performance Liquid Chromatography (HPLC) method was developed, optimized, and validated for Linagliptin/Metformin assay. HPLC method is developed for the assay analysis of Linagliptin and Metformin HCl simultaneously; the active substances are 2.5 mg and 1000mg respectively in the film-coated commercial tablet. The concentration range is validated for 0.00025 mg/ml – 0.0025 mg/ml for Linagliptin and 0.1 mg/ml - 1 mg/ml for metformin HCl. Specificity, linearity, precision, intermediate precision, accuracy, and robustness parameters were tested and this proposed method was found to be specific, precise, and robust.

Keywords: Linagliptin; Metformin; HPLC; Validation

Introduction

Linagliptin and metformin are two antidiabetic drugs that are often used in combination for therapy to manage type 2 diabetes [1]. Linagliptin is a Dipeptidyl Peptidase-4 (DPP-4) inhibitor that increases the levels of incretin hormones, stimulating insulin secretion [2]. Metformin is a biguanide that reduces glucose production in the liver and improves insulin sensitivity [3]. Clinical trials have been conducted to evaluate the safety and efficacy of the combination therapy of linagliptin and metformin in patients with type 2 diabetes [4]. In a Phase 2 study, it was found that the combination therapy significantly improved glycemic control compared to either drug alone with no significant adverse events reported [5]. Several phase 3 clinical trials have evaluated the efficacy and safety of linagliptin and metformin combination therapy in patients with type 2 diabetes [6]. In a randomized, double-blind, placebo-controlled trial, the combination of linagliptin and metformin was shown to be superior to either medication alone in reducing HbA1c levels and improving glycemic control over 24 weeks of treatment [7].

However, like any medication, there are potential adverse effects associated with linagliptin and metformin [8]. Some common adverse effects of linagliptin include upper respiratory tract infections, headaches, and hypoglycemia [9]. Common adverse effects of metformin include gastrointestinal symptoms such as nausea, vomiting, and diarrhea [10]. Rare but serious adverse effects of metformin include lactic acidosis, which can be life-threatening [11].

Chromatographic methods have broad applications in both analytical and purification processes [12]. Determining the drug and its byproducts or impurities is crucial, as this information is significant from both the pharmacological and toxicological perspectives, particularly for the pharmaceutical industry and scientists [13]. To ensure safety during pharmaceutical development, it is necessary to establish monitoring techniques for stability tests on raw materials, impurities, and degradation products, due to their potential for toxicity [14]. When the method is straightforward and readily applicable, simultaneous or direct determinations are preferred [14,15]. A chemical component originating from the drug substance or synthesized during the process is considered an impurity in the drug mass [15]. The safety of the drug relies not only on the toxicity of the active substance but also on its pharmaceutical impurities [16]. It is crucial to detect and quantify impurities in raw pharmaceutical materials and degradation products that may form during formulation, manufacturing, or storage. These impurities, also known as "related substances," may have pharmacological or toxicological significance. As such, their presence and levels in products indicate product quality and can pose a risk to patient safety [17-19].

High-Performance Liquid Chromatography (HPLC) is a highly effective analytical method utilized for separating, identifying, and quantifying chemical compounds in diverse sample types [13]. Within pharmaceutical analysis, HPLC is extensively employed for determining drugs and their metabolites in biological fluids, including blood, urine, and plasma, as well as in pharmaceutical formulations [20].

Extensive validation of an HPLC method is essential as it is commonly used to regularly analyze raw materials or drugs during various stages, including material acceptance, preformulation, quality control, and storage [21-23].

We present a rapid and cost-effective HPLC analytical technique to measure the quantities of linagliptin and metformin in pharmaceutical dosage forms or raw materials. The method has been validated by the ICH guidelines on "Validation of Analytical Procedures: Text and Methodology [24].

We evaluated various parameters, including specificity, linearity, recovery, precision (system precision, method precision, and intermediate precision), robustness, solution stability, and system suitability, and found that all the results were within acceptable limits. Therefore, the method can be employed or adopted for other intended purposes.

Materials and Methods

Chemicals

Analytical grade water, Acetonitrile (Isolab, Istanbul, Turkey), Potassium hydroxide (Isolab, Istanbul, Turkey), and Potassium Dihydrogen Phosphate (Merck, Merck KGaA, Darmstadt, Germany) were used.). Other chemicals and ingredients were of analytical grade. Linagliptin/Metformin (2.5/1000) film-coated tablets were obtained from the market.

HPLC

Zorbax SB CN (250mm x 4.6 mm, 5μm) (CA, USA) was used for the separation of compounds in HPLC (Shimadzu, Maryland, USA) analysis with a photodiode array detector. The separation was carried out using an isocratic elution, meaning that the mobile phase composition remained constant throughout the run time. The flow rate was set at 1.0 ml/min, and the column temperature was kept at 25°C. The photodiode array detector was used for the analytical detection of the separated compounds at a wavelength of 265 nm. The run time for the entire separation and analysis process was 8 minutes.

Assay

HPLC method is used for the assay analysis of Linagliptin and Metformin HCl (2.5 mg / 1000mg) in film-coated tablets obtained from the market. The concentration range was 0.00025 mg/ml – 0.0025 mg/ml for Linagliptin and 0.1 mg/ml - 1 mg/ml for metformin HCl. Specificity linearity, precision, intermediate precision, accuracy, and robustness parameters were tested in the report. Phosphate Buffer / Acetonitrile mixture (65:35) was used as a mobile phase. 10μl of solution was injected into the chromatographic system several times (3 times). System compatibility was checked with consecutive runs/readings of the standard solution.

Preparation of Samples from Tablets

10 tablets are weighed. The average weight tablets are crushed and the film tablet powder equivalent to 2.5 mg Linagliptin is weighed into a 200 ml volumetric flask, dissolving by holding it for 10 minutes with the help of an ultrasonic bath and completed with a dilution solution to the volume. 10 ml of this solution is taken to a 100 ml volumetric flask and completed to the volume with dilution solution. It is filtered through a 0.45 μm pore-sized filter and the vial was sealed. (C _Linagliptin: 0.00125 mg/mL), (C _{Metformin HCI}: 0.5 mg/mL)

Linagliptin%:

A_t: Active ingredient peak area obtained from the test solution

A_s: Active ingredient peak area obtained from the standard solution

W_s: Standard Weighing (mg)

P: as is the potency of standard

M_w: Average weight of tablet (mg)

W_t: Samples weight (mg)

T: Theoretical label content of tablets (2.5 mg)

Metformin HCI%:

A_t: Active ingredient peak area obtained from the test solution

A_s: Active ingredient peak area obtained from the standard solution

W_s: Standard Weighing (mg)

P: Potency of standard

M_w: Average weight of tablet (mg)

W_t: Samples weight (mg)

T: Theoretical label content of tablets (1000 mg)

Standard Stock Solution

25 mg of linagliptin working standard is taken into a 200 mL volumetric flask, dissolved by holding it for 10 minutes in the ultrasonic bath, and completed to its volume with dilution solution.

(C _Linagliptin : 0,125 mg/mL)

Standard Solution

100 mg of Metformin HCl working standard is taken into a 200 mL volumetric flask, dissolved by holding it for 10 minutes with the help of an ultrasonic bath, and 2 ml of the stock standard solution is added and the volume is completed with the dilution solution. It is filtered through a 0.45 micron RC filter and dialed.

(C _Linagliptin : 0.00125 mg/mL), (C _{Metformin HCI} : 0.5 mg/mL)

Placebo Solution:

Approximately 268.5 mg placebo is weighed into a 200 ml volumetric flask, some dilution solution is added, kept in an ultrasonic bath for 30 minutes, and completed with dilution solution to its volume. 10 ml of this solution is taken to a 100 ml volumetric flask and completed to the volume with dilution solution. It is filtered through the 0.45-micron filter and vialled.

Validation Parameters

The Linagliptin/Metformin assay on tablets was performed by ICH guidelines for the validation. The parameters of system suitability, linearity, specificity, recovery, precision, (system, method, and intermediate precision), robustness, and solution stability were tested [16].

Specificity

Specificity is the unequivocal determination of the substance to be analyzed in the product in the presence of matrix effects and additives [13]. The mobile phase, placebo, standard and sample solutions are prepared and analyzed as in the method.

There must be no interference with the active substance from the mobile phase, dilution solution, and placebo. The retention time of Linagliptin is 4.39 minutes and for Metformin HCl is 2.59 minutes.

Linearity

The linearity of the analytical method is that the test results obtained within the specified interval have the correct proportion to the concentration of the relevant substance and/or active substance in the sample. 20%, 50%, 80%, 100%, and 200% standard solutions are prepared for Linagliptin and Metformin HCI, and each concentration is injected and analyzed three times separately. The correlation coefficient was then determined. The correlation coefficient is represented as r2 (it should be >0.995). RSD% value of the peak areas for each concentration should be =1.0. The intercept value should be less than 5% of the average area [13].

Accuracy

The accuracy of the analytical method is expressed as the suitability of the prepared concentration of the substance to be analyzed in the sample and the concentration of the determined result of the assay The accuracy study will be prepared by adding the active substance to the placebo and analyzed as specified in the method, test solutions at 50% 100% and 200% levels and 3 from each level. % Recovery was then calculated. % Recovery should be 98.0% - 102.0%. RSD% of the calculated results from the recovery studies should be =1.0 [13,15].

Precision

The precision of an analytical method is the amount of scattering in the results obtained from multiple analyses of a homogeneous sample [25]. System precisions, repeatability, and intermediate precisions were evaluated.

System Precision

System precision is the measure of system performance independent of errors from the sample preparation phase. 6 consecutive injections are made from the standard solution, and the area values are determined. The mean of the results, Standard Deviation (SD), and percentage Relative Standard Deviation (% RSD) are calculated. The RSD value must not be greater than 2.0%.

Intermediate Precision

To see the effect of random changes during the day in the laboratory on the precision of the analytical method; it is tested by performing the same analysis with different columns and different analysts. Different analysts prepare samples defined with method precision. It is injected into the system using a different HPLC column system. The result is calculated for each sample. The % RSD between the results of 12 samples of two analysts is calculated. The RSD should be =2.0 % (n=12) [13,14].

Repeatability

It is the evaluation of the system performance within the errors that will come from the sample preparation stage [16]. The repeatability of the analytical method for assay analysis is tested using measurement data from 6 original samples prepared separately. Metformin's and Linagliptin's sample concentrations were adjusted to 0.5 mg/ml and 0.001251 mg/ml, respectively. The RSD of the results should be =2.0 %.

Robustness

The robustness of an analytical method is the measure that the Method parameters remain unaffected by known small changes and is an indication of the reliability of the analytical method in its normal use [16]. The following changes will be made in the method parameters and the effects of these changes on the system suitability parameters will be examined.

The Effect of the Temperature

Column temperature will be changed by 5°C (20°C – 25°C – 30 °C). The standard and test solution prepared as described in the sample preparation section is injected into the system and its compliance with the acceptance criteria is checked. The results are calculated and compared with the results obtained under the conditions of the method.

The Effect of Flow Rate

The flow rate will be changed to 0.2 ± ml /min (0.8 ml/min. – 1.0 ml/min. – 1.2 ml/min.). The standard and test solution prepared as described in the sample preparation section is injected into the system and its compliance with the acceptance criteria is checked. The results are calculated and compared with the results obtained under the conditions of the method. The difference between the original method and the modified method results must be =5.0%.

System Suitability

Six consecutive injections were done and the relative standard deviation between consecutive standard injections must be a maximum of 2.0%, the tailing factor should not exceed 1%. The proposed method was also tested for detection and quantification limits. The Limit of Detection (LOD) and Limit of Quantification (LOQ) of the procedure were also calculated according to the 3 s/m and 10 s/m criterion, respectively, where s is the standard deviation of the peak area ratios (n=4) of the sample and m is the slope of the corresponding calibration curve.

Stability of the Working Solutions

The retention period of the substance to be analyzed should be determined stably under the analysis conditions. The holding time to be selected is specific to the analysis. If the active substance degrades within this period and storage conditions, these conditions must be limited and specified in the method. Standard and sample solutions will be prepared according to the method, kept at chromatographic system temperature (5°C) for 24 hours, and analyzed. RSD of the results should be =1.0.

Results and Discussion

The retention time of metformin was 2.608 minutes and it was 4.417 minutes for linagliptin (Figure 1). Different columns with different polarities were tried. The system for standard solutions was accepted as suitable because the relative standard deviation between readings did not exceed 1%, tailing factors were less than 1.8, and the theoretical plate number was higher than 5000.