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Document Type : Original Article

Authors

Department of Chemistry, Vel Tech Rangarajan Dr. Sagunthala R&D Institute of Science and Technology, Avadi, Chennai-600062, India

Abstract

It has been developed and validated an accurate, sensitive, precise, quick, and gradient reverse phase HPLC (RP-HPLC) technique for combined quantification of Metformin, Linagliptin, and Empagliflozin in raw materials and pharmaceutical combined tablets. With acetonitrile as the organic solvent, the best separation was achieved on a 250 mm x 4.6 mm i.d., 5µ-particle size Inertsil®-Octadecyl-silyl-3V-Reverse-Phase-C18-column with acetonitrile as the non-polar-modifier and Dipotassium mono-hydrogen phosphate buffers system [0.03M] in water with pH 3.0 adjusted with ortho-phosphoric acid (0.1 %v/v) in the gradient mode of elution as an elution solvent at a speed of 1.0 mL.min−1. UV detection was at 230-nm. Metformin had a retention time of 2.421 minutes, Linagliptin's was 8.187 minutes, and Empagliflozin's was 11.71 minutes. With a correlation coefficient of about 0.9999, the peak-response was obtained as a function of concentration over the range of 80-480 mcg/mL for metformin, 0.4-2.4 mcg/mL for linagliptin, and 0.8-4.8 mcg/mL for empagliflozin. Metformin, Linagliptin, and Empagliflozin were shown to have a percentage assay of 99.92, 99.72, and 99.74, respectively. Metformin, Linagliptin, and Empagliflozin, each have a limit of detection of 0.04 g/mL, 0.004 g/mL, and 0.004 g/mL, respectively. Metformin, Linagliptin, and Empagliflozin, each have a limit of quantification (LOQ) of 0.12 ng/mL, 0.012 g/mL, and 0.012 g/mL, respectively. The presence of excipients in the formulation had no effect on the assay method. The procedure is appropriate for usage in QC- laboratories since it is quick and precise.

Graphical Abstract

RP-LC Method in Gradient mode for Combined Quantification of Metformin, Linagliptin, and Empagliflozin in Combination Tablets

Keywords

Main Subjects

Introduction

Empagliflozin inhibits the Na-glucose-co-transporter system that can be taken orally (SGLT2). The molecular weight is 450.91D and the chemical formula is C23H27ClO7. Empagliflozin is a non-hygroscopic white to yellowish powder [1-3]. It's soluble in water, C2H5OH, and acetonitrile; it’s soluble in 1:1 proportion of CH3CN-H2O; and essentially not soluble in toluene4. Linagliptin is given by mouth, which inhibits the di-peptidyl-peptidase-4 (DPP-4) [4]. It is soluble in water, methanol, ethanol, and acetonitrile, as well as soluble in 50 percent acetonitrile/water, and essentially insoluble in toluene4 Linagliptin is an orally administered dipeptidyl peptidase-4 (DPP-4) inhibitor. The chemical formula is C25-H28-N8-O2, and the molecular weight is 472.54. Linagliptin is soluble in methanol, ethanol very sparingly, isopropanol only very faintly, and acetone only very weakly. Metformin-HCl is a crystalline white-to-off-white powder with a molecular weight of 165.63D and a molecular formula of C4-H11-N5•HCl. Metformin-HCl is soluble in water but almost insoluble in acetone, pet-ether, and chloroform. The pKa-of metformin is 12.4 and metformin hydrochloride in a 1% aqueous solution has a pH of 6.68. Trijardy® is a novel FDC for diabetes therapy which combines extended-release metformin (metformin-XR) with empagliflozin, an SGLT2 inhibitor, and linagliptin, a DPP-4 inhibitor. Each TRIJARDY XR film coated tablet contains an extended-release metformin-HCl central tablet covered with the empagliflozin as fast-released pharmacological ingredient. In the literature, RP-HPLC approach has been published for measuring the potency of metformin and other oral anti-diabetic drugs [5-8]. Nevertheless, no techniques for combined determination of metformin-HCl-with-linagliptin & empagliflozin have been published in oral fixed dosage form. Furthermore, no official or preliminary monograph on this combination of analytes has been published in any of the compendial pharmacopoeias [9]. The goal of this study was to develop an accurate and efficient RP-HPLC method to estimate a new combination of antidiabetic drugs in fixed dosage forms for oral administration. The validation of the devised approach is also addressed in this study, as per ICH standards [12].

Martials and Methods

  • Metformin, 99%, Linagliptin of 99% and Empagliflozin of 99% pure are acquired from Sigma-Aldrich Chemicals, Mumbai, India.
  • Rankem-Fine-Chemicals of HPLC- Grade- Acetonitrile.
  • Rankem-Fine-Chemicals AR_grade: DiPotassium-monohydrogen-phosphate (dibasic, K2HPO4) [0.03M].
  • Ortho-H3PO3, 85% (v/v) obtained from Quligen-Fine chemicals.
  • Chromatographic-Grade water.

Chromatographic-Instrument

Quantitative HPLC was carried out on a Shimadzu Prominence high-performance liquid chromatograph with a PDA detector module, which included an automated injector with a 20-l injection volume and a quadra-pump. The utilized column was a Reverse-Phase-Inertsil-Octa-Decyl-S-3V-C18 column (250 mm x 4.6 mm internal diameter with particle size 5 µm). Spin-chrome Software was installed on the HPLC equipment. The column temperature 40 °C was adjusted and eluted over 20.0 minutes at a mobile solvent speed of 1.0 mL.min-1 under linear gradient conditions. The organic modifier is acetonitrile, while the mobile phase is water with Dipotassium hydrogen phosphate [0.03M] and a pH of 3.0 adjusted with ortho-phosphoric acid (0.1 percent v/v) (0.1% v/v). It was degassed and filtered via 0.45 m Nylon membrane filters before use. For three analytes, UV detection at 230 nm was used as isobestic wavelength detection with a PDA detector.

Preparation of Primary Standard Drug Solutions

To make the primary standard stock solution, 400mg of Metformin, 2 mg of Linagliptin, and 4 mg of Empagliflozin were dissolved in a volumetric flask (100 mL) with 20 mL of diluent (1:1 v/v CH3CN:H2O), sonicated for 15 minutes, and then brought up to 100mL with diluents to get the primary standard stock solution containing 4000 µg.mL-1 of Metformin, 20 µg.mL-1 of Linagliptin and 40 µg.mL-1 of Empagliflozin.

 Preparation of Working Standard Drug Solution

In a 50 mL volumetric flask containing 400 µg.mL-1 Metformin, 2µg.mL-1 Linagliptin, and 4 µg.mL-1 Empagliflozin, the working standard solution was produced up to 50 mL with diluent (1:1 v/v CH3CN:H2O) from 5 mL of the above-mentioned primary-stock solution.

Sample Preparation

Twenty Trijardy-XR® combination tablets, which contain an extended-release metformin-HCl central tablet covered by the empagliflozin and linagliptin as fast-released pharmacological ingredient components are available in 10 mg empagliflozin/5 mg linagliptin/1000 mg metformin-HCl extended-release combined tablet dosage forms, were accurately weighed, and the mean weight was determined, as well. An equivalent-quantity of the homogeneous powder equivalent to 400 mg metformin, 4 mg empagliflozin, and 2 mg linagliptin was transferred, and 50 mL 0.02 M KH2PO4 and 0.02 M K2HPO4 added. CH3OH:H2O at ratio of 90:10 (v/v) was added as diluent, sonicated for 30 minutes, diluted to 100 mL and pH adjusted to 7.0 with ortho-H3PO3 in water. The resulting solution was miscible completely by sonication for 30 min, diluted up to 100 mL with ortho-phosphoric acid. Metformin 400 µg.mL-1, Linagliptin 2 µg mL-1, and Empagliflozin 4 µg mL-1 are all present in the sample solution. The individual linear regression equations were used to determine the amounts of metformin, empagliflozin, and linagliptin in teritiary mixture of sample solution form fixed-dosage-forms.

Linearity

Aliquots of Metformin, Linagliptin, and Empagliflozin working stock solutions were placed in various 10mL volumetric flasks and made the volume up to the 10mL with the mobile phase, yielding in final strengths of 80-480    µg.mL-1, 0.4-2.4 µg.mL-1, and 0.8-4.8 µg.mL-1, respectively (Table 1). The peak areas and retention times of each of these drug solutions (loaded at 10 µL) were measured three times in the column. Using a PDA-detector set at 230 nm, a linearity-graph was generated by plotting peak areas versus Metformin, Linagliptin, and Empagliflozin concentrations in µg-mL-1.

Accuracy

The approach’s accuracy was found by evaluating the drugs’ recovery by using the standard-spiking method. To assess if the analytes contained in the formulation caused positive or negative interventions, known amounts of each drug equivalent to 10 percent standard drug solution were added to 80 percent, 100 percent, and 120 percent of the target test concentrations a formulation mixture. Each set-of-addition was replicated three times at each dilution level. The results are compared to a competent reference standard after extraction of sample preparation. The percentage of analytes re-covered by the assay was used to assess the accuracy. Table 4 shows the results of accuracy investigations on standard solution and process-related impurity; the recovery measurements suggest that the procedure was accurate.

Precision

Quality-control samples in 100 % (w/v) dilution were used to assess intraday and interday precision. On the same day, six replicates of the target concentrations were examined for intra-day variation, and six replicates were examined for inter-day variation on three different days. The method’s repeatability is indicated by the low RSD value (1%) (Table 4).

Limits of Detection and Quantification

The method’s LOD was set at the lowest concentrations of active pharmaceutical components with a signal-to-noise (S/N) ratio of around 3 (LOD). The lowest active therapeutic medication concentrations which can be assessed with acceptable precision and accuracy while maintaining a signal-to-noise (S/N) ratio of roughly 10 (LOQ).

Method Applicability

Our study group evaluated the newly created method by applying it to pharmaceutical tablets for estimating Metformin, Linagliptin, and Empagliflozin.

Method Validation Tests

Method precision (RSD, percent), method accuracy (recovery percent & %RSD), linear range (r2), and LOD & LOQ were explored as recommended method validation characteristics.

Result and Discussion

Linearity

With a correlation coefficient of 1.0, the graph of chromatographic-peak areas of all analytes versus respective concentrations was indicated to be linear in the band of 80-480 µg. mL-1 for Metformin, 0.4-2.4 µg.mL-1 for Linagliptin, and 0.8-4.8 µg.mL-1 for Empagliflozin (Table 1). The least square fit data of linear regression analysis was derived from the measurements given in Table 2. Metformin’s regression formula is y = 1E+06x, Linagliptin's is y=122817x, and Empagliflozin's is y = 187710x. Table 2 presents the regression parameters for this technique that include slope, intercept, and % RSD. These findings suggest that there was a significant correlation.

Table 1: Evaluation of precision with-in-day and day-to-day analysis

With-in-day Precision study of 100% dilution containing Metformin (400 µg/Ml), Linagliptin (2 µg/mL) and Empaglifozin (4 µg/mL)

Day-to-Day Precision study of 100% dilution containing –Metformin (400 µg/mL), Linagliptin (2 µg/mL) and Empaglifozin (4 µg/mL)

Metformin

Linagliptin

Empagliflozin

Metformin

Linagliptin

Empagliflozin

S. No

R.T

Peak-Area

R.T

Peak-Area

R.T

Peak-Area

R.T

Peak-Area

R.T

Peak- Area

R.T

926267

1

2.421

5735858

8.187

622809

11.716

935541

2.425

5771972

8.194

607055

11.693

927709

2

2.422

5738972

8.189

606552

11.713

922964

2.426

5773228

8.195

608105

11.696

928509

3

2.422

5753564

8.189

605189

11.702

921717

2.426

5778001

8.196

608723

11.692

929952

4

2.424

5743597

8.191

605106

11.700

922997

2.426

5779250

8.200

609552

11.699

930743

5

2.425

5747941

8.194

605885

11.704

923517

2.426

5784303

8.198

610453

11.693

931079

6

2.425

5751030

8.194

606724

11.698

923612

2.426

5792709

8.193

610354

11.685

929043.2

Average

2.423

5745160.3

8.191

608710.8

11.705

925058

2.426

5779910.5

8.196

609040.3

11.693

1877.9

Std. Dev

0.0017

6929.4

0.0028

6939.0

0.0073

5179.8

0.0042

7679.2

0.0024

1334.2

0.0046

0.2

% RSD

0.060

0.1

0.033

1.1

0.061

0.6

0.019

0.1

0.031

0.2

0.040

926267

 

Table 2: Regression analysis & Operating-System Suitability Results

Study-Parameter

Metformin

Linagliptin

Empagliflozin

Retention Time (min)

2.421

8.187

11.716

Tailing Factor

1.454

1.399

1.090

Peak areas

5735858

622809

935541

Percentage of peak areas

78.65

8.53

12.82

Theoretical Plates

3033.879

17953.186

31545.673

Resolution

0.000

27.439

13.884

Linear range in (µg/mL)

80-480

0.4-2.4

0.8-4.8

Limit-of-Detection  (µg.mL-1)

0.04

0.004

0.004

Limit-of-Quantification (µg.mL-1)

0.12

0.012

0.012

Correlation-Coefficient (r2)

0.998

0.998

0.998

Assay-in-Percentage (%)

99.92

99.32

99.74

 

Accuracy

Individual recovery of analyte at 80 %-dilution level on w/v basis, 100 %-dilution level on w/v basis and 120 %-dilution level on w/v basis of prescribed concentrations was 98.1 percent to 102.45 percent, demonstrating the method’s accuracy. The RSD was usually less than 1% in these data, demonstrating that the technique seems to be very accurate and generates consistent results (Table 3).

 

Table 3: Summery of the standard calibration Curve for Linearity experiment

Amount-of Metformin (µg/mL)

Chromatogram

Amount-of  Linagliptin (µg/mL)

Chromatogram

Amount-of  Empagliflozin in µg/mL

Chromatogram

80

Peak- Area

0.4

Peak- Area

0.8

Peak- Area

160

1196011

0.80

124271

1.6

189169

240

2351071

1.20

244392

2.4

370631

320

3525071

1.60

373497

3.2

562549

400

4690599

2

492631

4.0

750593

480

5756301

2.4

609699

4.8

939968

 

Precision

Table 4 summarizes the intra-day and inter-day fluctuation in precision analysis. The method’s repeatability is indicated by the low RSD value (lessthan-1%). These results indicate that the approach has a high level of precision and repeatability, both within a single analytical run and across multiple runs (Table 4).

 

Table 4: Accuracy evaluation by Spike-analysis method

Accuracy parameter

Standard-addition with 80%w/v of the Working Standard

Standard-addition with 100%w/v of the Working Standard

Standard-addition with 120% (w/v) of the Working Standard

Empagliflozin

 (3.2 µg/mL)

Linagliptin

 (1.60 µg/mL)

Metformin

(320 µg/mL)

Empagliflozin

(4.0 µg/mL)

Linagliptin

(2.0 µg/mL)

Metformin

(400 µg/mL)

Empagliflozin

(4.8 µg/mL)

Linagliptin

(2.4 µg/mL)

Metformin

(480 µg/mL)

Amount added

0.40

0.20

40

0.4

0.2

80

0.4

0.2

80

Amount Found

3.57

1.78

358

4.36

2.19

479

5.22

2.41

558

% Recovery

99.16

98.88

99.44

99.09

99.54

99.79

100.2

99.58

99.64

% RSD.

0.02

0.04

0.15

0.28

0.54

0.22

0.62

0.74

0.82

 

Robustness

At three different levels, –ve2, 0, and +ve2, the influence of minute but intentional alterations in the separation parameters was explored. The experimental settings were purposely changed at three distinct levels to determine the robustness of this approach, and the RT and chromatographic responses were evaluated. The effect was observed by modifying one factor at a time. The RT and the method response were not affected by changing the stationary phase, pH of elution solvents by 0.2 units (pH=3.2 and pH=2.8), or mobile phase flow rate by 1.0 mL.min-1 (0.8 and 1.2 mL.min-1, demonstrating that the procedure was robust. Table 5 summarises the findings.

 

 Table 5: The method’s Robustness can be tested by changing the chromatographic settings (n=3, 100%w/v Working dilution contains Metformin-400 µg/mL, Linagliptin- 2 µg/mL and Empagliflozin-4 µg/mL)

S.No.

Study of Peak areas with Different HPLC Column

Study of Peak areas with Mobile Phase pH increase

Study of Peak areas with Mobile Phase pH decrease

Metformin

Linagliptin

Empagliflozin

Metformin

Linagliptin

Empagliflozin

Metformin

Linagliptin

Empagliflozin

1

6035770

633834

971248

5658854

545876

837626

5289148

639916

979780

2

6045001

634525

971486

5661336

542994

828083

5298146

641478

980233

3

6047370

635116

972876

5660917

548447

826794

5302371

642333

981138

Mean

6042713

634492

971870

5660369

545772

830834

5296555

641242

980384

% RSD

0.101

0.101

0.090

0.023

0.500

0.712

0.128

0.191

0.070

S.No.

Study of Retention times with Different HPLC Column

Study of Retention times with Mobile Phase pH increase

Study of Retention times with Mobile Phase pH decrease

Metformin

Linagliptin

Empagliflozin

Metformin

Linagliptin

Empagliflozin

Metformin

Linagliptin

Empagliflozin

1

2.420

8.166

11.732

2.257

7.943

10.973

2.345

8.128

11.718

2

2.417

8.162

11.722

2.259

7.937

10.972

2.347

8.126

11.713

3

2.418

8.163

11.728

2.257

7.930

10.970

2.347

8.124

11.718

Mean

2.418

8.164

11.728

2.257

7.937

10.972

2.346

8.126

11.716

% RSD

0.044

0.029

0.044

0.041

0.080

0.016

0.061

0.022

0.027

 

Limit-of-Detection & Limit-of-Quantifications

The limit-of-detection (LOD) for Metformin is 0.04µg/mL, and for Linagliptin and Empagliflozin was found to be 0.004 µg.mL-1. The limit-of-quantification (LOQ) for Metformin is 0.12 µg.mL-1, and for Linagliptin and Empagliflozin was found to be 0.012 µg.mL-1. These numbers illustrate the method’s high sensitivity, which is essential in most investigations, as well as the fact that it can be used to detect and quantify analytes over a wide concentration range.

Specificity

The RTs for Metformin, Linagliptin, and empagliflozin were determined to be 2.42 minutes for Metformin, 8.18 minutes for Linagliptin, and 11.72 minutes for empagliflozin, according to the representative chromatogram given in Figure 1. When the pharmaceutical tablet matrices were evaluated, no indication of excipient interference signals was observed in the respective RTs of the chromatogram. It indicates that the analytes were not disturbing the probable merging peaks. As a result, this technique can be employed with certainty.

Figure 1: Chromatogram of Metformin-400µg/mL, Linagliptin-2µg/mL and Empagliflozin- 4µg/mL analyzed by the optimized Gradient RP-HPLC method

Figure 2: Linearity graphs of metformin for dilution of standard solutions

Figure 3: Linearity graphs of Linagliptin for dilution of standard solutions

Figure 4: Linearity graphs of empagliflozin for dilution of standard solutions

A gradient RP- HPLC procedure for assaying the active ingredients was developed due to lack of an easy, reproducible, and quick-to-use method for the determination of Metformin, Linagliptin, and Empagliflozin concentrations in formulary matrices. We examined the effect of various HPLC technique variables on the final result of the study to optimize the chromatographic parameters, various proportions of CH3OH-H2O, CH3CN-H2O, and CH3CN-KH2PO4 buffer were tested. After several early investigatory tests, CH3CN-KH2PO4 buffer system [pH 3.0], it was chosen over other mobile phases because it resulted in improved resolution of pharmacologically active components. This procedure gives the good separation of analytes after multiple exploratory & investigatory trail runs. All three active pharmaceutical analytes had an excellent UV sensitivity and were interference-free at 230 nm. The analyte peaks were highly defined and without any incidence of tailing under these conditions. The aforementioned set of conditions were chosen for additional validation after considering the entire body of data acquired from this extensive study.

Conclusion

In this study, an efficient and commonly available HPLC method was devised for analyzing Metformin, Linagliptin, and Empagliflozin in pharmaceutical matrices. This method’s key advantages are its significantly reduced run times, ease of use, and of operation. All of these features are critical in operation, especially when analyzing a large number of samples. The validation experiments demonstrated that the procedural approach has a large calibration concentration range, adequate precision, accuracy, and practically reliable sensitivity. The method can be used for regular analysis in formulation QC-studies and allows for a straightforward, selective, sensitive, and specific assessment of Metformin, Linagliptin, and Empagliflozin.

Acknowledgments

The authors are grateful to the Management of Vel Tech University and commercial organization of Vishnu Chemicals Ltd. Hyderabad for their encouragement and providing the necessary facilities to carry out the entry research work.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

 

Authors' contributions

All authors contributed toward data analysis, drafting and revising the paper and agreed to responsible for all the aspects of this work.

 

Conflict of Interest

We have no conflicts of interest to disclose.

 

ORCID:

Hazarathaiah Yadav

https://orcid.org/0000-0002-5124-879X

Mallibabu

https://orcid.org/0000-0001-8597-9606

HOW TO CITE THIS ARTICLE

C. Hazarathaiah Yadav, A. Mallibabu. RP-LC Method in Gradient mode for Combined Quantification of Metformin, Linagliptin, and Empagliflozin in Combination Tablets, J. Med. Chem. Sci., 2022, 5(6) 1018-1025

https://doi.org/10.26655/JMCHEMSCI.2022.6.15

URL: http://www.jmchemsci.com/article_149694.html    

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