Review on Analytical Methods for Estimation of Antidiabetic Drugs: Empagliflozin, Linagliptin and Metformin Hydrochloride

 

M. M. Eswarudu*, G. Ouchitya, N. Sudhakar Reddy, M. Deekshitha, P. Srinivasa Babu

Department of Pharmaceutical Analysis, Vignan Pharmacy College, Vadlamudi, 522213, Andhra Pradesh, India

*Corresponding Author E-mail: eswarmunnangi@gmail.com

 

ABSTRACT:

Pharmaceutical analysis is necessary at all stages of the drug development process, including formulation development, stability studies, and quality control. It is also used for characterizing the composition of different dosage forms in quantitative and qualitative ways. Comprehensive literature survey forms the foundation stone for the focused analysis of research activity. This review article represents the collection and discussion of various analytical methods available in the literature for the determination of oral anti-diabetic drugs like Empagliflozin (EMPA), Linagliptin (LINA) and Metformin hydrochloride (MET) for the treatment of type II diabetes mellitus, consisting of UV, TLC, HPTLC, and HPLC. The anticipated review provides details about the comparative utilization of various analytical techniques for the determination of EMPA, LINA and MET. This review article can be effectively explored to conduct future analytical investigation for the estimation of selected drugs in pharmaceutical and biological samples.

 

KEYWORDS: Empagliflozin, Linagliptin, Metformin hydrochloride, HPTLC, RP-HPLC.

 

 

INTRODUCTION:

Empagliflozin is an orally active sodium glucose co-transporter-2 (SGLT-2) inhibitor used to control type II diabetes. It belongs to gliflozin class with chemical name (2S,3R,4R,5S,6R)-2-[4-chloro-3-[[4-[(3S)-oxolan-3-yl] oxyphenyl] methyl] phenyl]-6-(hydroxymethyl) oxane-3,4,5-triol and having an empirical formula and molecular weight of C23H27ClO7 and 450.91g/mol. It is very slightly soluble in water, slightly soluble in acetonitrile and ethanol and sparingly soluble in methanol1. Chemical Structure of Empagliflozin is shown in Figure-1.

 

Figure-1: Chemical Structure of Empagliflozin

 

Linagliptin is an orally-active inhibitor of the dipeptidyl peptidase-4 (DPP-4) enzyme. Chemically Linagliptin is 8-[(3R)-3-aminopiperidin-1-yl]-7-but-2-ynyl-3-methyl-1-[(4-methylquinazolin-2-yl) methyl] purine-2,6-dione having an empirical formula of C25H28N8O2 with molecular weight 472.5422 g/mol. It is soluble in methanol, sparingly soluble in ethanol, and very slightly soluble in isopropanol2. Chemical structure of Linagliptin is depicted in Figure-2.

 

Figure-2: Chemical Structure of Linagliptin

 

Metformin hydrochloride belongs to antihyperglycemic agent of the biguanide class which is used for the management of type II diabetes. For the purpose of increasing solubility, metformin formulations are present in its salt form as Metformin Hydrochloride.  Chemically Metformin hydrochloride is 3-(diamino methylidene)-1,1-dimethylguanidine having an empirical formula of C14H11N5.HCl with molecular weight 165.625g/mole. It is freely soluble in water, slightly soluble in alcohol and acetonitrile and practically insoluble in acetone, ether and chloroform3. Metformin hydrochloride structure is shown in Figure-3.

 

Empagliflozin, Linagliptin and Metformin hydrochloride combination was approved for use in the United States in January 2020. The combination works by three complementary mechanisms which help in managing blood glucose in adult with type II diabetes mellitus. The fixed dose combination of these three drugs was sold under the brand name Trijardy XR4. Oral Empagliflozin, Linagliptin and Metformin hydrochloride combination of controlled release tablets as a triple fixed-dose combination (FDC), are being developed by Boehringer Ingelheimin collaboration with Eli Lilly, for the treatment of type II diabetes mellitus5.

 

Figure-3: Chemical Structure of Metformin Hydrochloride

 

Reported analytical methods:

Table –1: Methods for Empagliflozin, Linagliptin and Metformin Hydrochloride combination

S. No.

Method

Description

Ref. No.

1

RP-HPLC

Stationary Phase: Pheonex C18 column (250mm X 4.6mm ;5µm)

Mobile Phase: Acetonitrile: Methanol: Water (27:20:53 % v/v/v)

Wavelength: 223nm

Flow rate: 1ml/min

Linearity range: 0.5-5 µg/ml EMPA; 0.25-2.5µg/ml LIN; 50-500 µg/ml MET

Retention time: 14.5 min EMPA; 3.4 min LIN; 2.01 min MET

LOD: 0.03µg/ml EMPA; 0.04 µg/ml LIN; 5.65µg/ml MET

LOQ: 0.10µg/ml EMPA; 0.12µg/ml LIN; 17.12µg/ml MET

Theoretical plates: 2426.62 EMPA; 32021 LIN; 2238 MET

Tailing factor: 1.0 EMPA; 1.0 LIN; 1.25 MET

6

2

RP-HPLC

Stationary Phase: Kromosil (250mmX4.6mm;5µm) column

Mobile Phase: Buffer: Acetonitrile (45:55 %v/v)

Flow rate: 1ml/min

Linearity range: 2.5 – 15µg/ml EMPA; 250-1500µg/ml MET; 1.25-7.5 µg/ml LIN

Retention time: 2.787min EMPA; 3.419 min MET; 2.370 min LIN

%Recovery: 99.47 % EMPA; 100.01 % MET; 99.99 % LIN

LOD: 0.08µg/ml EMPA; 9.22µg/ml MET; 0.02µg/ml LIN

LOQ: 0.25µg/ml EMPA; 27.94µg/ml MET; 0.05µg/ml LIN

7

3

HPLC

Stationary Phase: C18 column

Mobile Phase: Acetonitrile: phosphate buffer (38:62 % v/v)

Wavelength: 222nm

Flow rate: 1ml/min

Retention time: 3.93 min EMPA; 2.04 min MET; 5.99 min LIN

Linearity range: 0.2-40µg/ml EMPA; 1-200µg/ml MET; 0.1-20µg/ml LIN

8

*EMPA-Empagliflozin, MET-Metformin, LIN-Linagliptin

 

Table – 2: Methods for Empagliflozin and Linagliptin combination

S. No.

Method

Description

Ref. No.

1

RP-HPLC

Stationary Phase: C18 column (BDS 250 mm X4.6 mm ;5 µm)

Mobile Phase: 0.1% Perchloric acid: Acetonitrile (60:40)

Wavelength: 230nm

Flow rate: 1ml/min

Retention time: 2.05 min EMPA; 4.10 min LIN

Linearity range: 25 -150µg/ml EMPA; 12.5 -75µg/ml LIN

% Recovery: 100.96 -101.48 % EMPA; 100.09 -101.13 % LIN

LOD: 0.03µg/ml EMPA; 0.43µg/ml LIN

LOQ: 0.09µg/ml EMPA ;1.32µg/ml LIN

9

2

RP-HPLC

Stationary Phase: Thermo C18 column (250 mm X4.6 mm ;5 µm)

Mobile Phase: Acetonitrile: Methanol (50:50 %v/v)

Wavelength: 280nm; Flow rate: 1ml/min

Linearity range: 5-25µg/ml EMPA; 1-5µg/ml LIN

Run time: 7 min

Retention time: 3.3 min EMPA; 4.08 min LIN

LOD:  0.12µg/ml EMPA; 0.10µg/ml LIN; LOQ: 0.40µg/ml EMPA; 0.25µg/ml LIN

Theoretical plates: 2639.83 EMPA; 3169.83LIN

10

3

RP-HPLC

Stationary Phase: Kromosil (250 mmX4.6 mm ;5 µm) column

Mobile Phase: 0.1% Ortho phosphoric acid buffer: Acetonitrile (60:40 %v/v)

Wavelength: 230nm; Flow rate: 1 mi/min

Retention time: 2.137 min EMPA; 2.759 min LIN

LOD: 0.70µg/ml EMPA; 0.23µg/ml LIN; LOQ: 1.34µg/ml EMPA; 0.44µg/ml LIN

11

4

TLC

Stationary Phase: TLC Silica gel 60 F254 aluminium plates (20 cm X10 cm; 0.25 mm)

Mobile Phase:  Chloroform: Methanol: Ammonia (9:1:0.1v/v)

Linearity range: 0.4-10µg/band EMPA; 0.2-5.0µg/band LIN 

Rf value: 0.25 EMPA; 0.56 LIN

12

5

HPTLC

Stationary Phase: HPTLC Nano silica gel 60 F254 glass plates (10 cm X10 cm; 0.1 mm)

Mobile Phase:  Chloroform: Methanol: Ammonia (25%) (10:1:0.1v/v)

Linearity range: 0.1-5µg/band EMPA; 0.05-2.5µg/band LIN 

Rf values: 0.31 EMPA; 0.71 LIN

13

6

HPTLC

Linearity range: 0.2-1.2 µg/band EMPA; 0.1-0.6 µg/band LIN

LOD: 1.678µg/band EMPA; 1.56µg/band LIN

LOQ: 1.565µg/band EMPA; 1.46µg/band LIN

% Recovery: 99.71 % EMPA; 99.64 % LIN

Rf value: 0.57 EMPA; 0.22 LIN

14

7

UV

Wavelength: 276nm EMPA; 293nm LIN

Linearity range: 5-80µg/ml (both)

% Recovery: 99-101 % (both)

LOD: 0.7099µg/ml EMPA; 0.7916µg/ml LIN

LOQ: 2.151µg/ml EMPA; 2.398µg/ml LIN

15

 

Table – 3: Methods for Empagliflozin and Metformin combination

S. No.

Method

Description

Ref. No.

1

RP-HPLC

Stationary Phase: Kromosil C18 column (50mm X4.6mm; 5µm)

Mobile Phase: Acetonitrile: 0.1 % Ortho phosphoric acid (50:50 % v/v)

Wavelength: 260 nm

Flow rate: 1ml/min

Linearity range: 1.25–7.50µg/ml EMPA; 125-750µg/ml MET

Run time: 6 min

Retention time: 3.20 min EMPA; 2.19 min MET

%Recovery: 100.52 % EMPA; 100.55 % MET

LOD: 0.01µg/ml EMPA; 0.50µg/ml MET

LOQ: 0.03µg/ml EMPA; 1.52µg/ml MET

16

2

HPLC

Stationary Phase: X – Select -HSS C18 SB (4.6 mmX25 cm ;5 µm)

Mobile Phase: Potassium dihydrogen phosphate: Acetonitrile

(60:40 v/v)

Wavelength: 255nm

Flow rate: 1mi/min

Retention time: 6.4 min EMPA; 2.6 min MET

Run time: 8 min

Injection volume: 10µl

Linearity range: 3.13-9.38 µg/ml EMPA;250-750 µg/ml MET

LOD: 0.352µg/ml EMPA; 36.80µg/ml MET

LOQ: 1.055µg/ml EMPA; 110.401µg/ml MET

Tailing factor: 6.37 EMPA; 2.66 MET

17

3

HPTLC

Stationary Phase: Silica gel precoated plates

Mobile Phase: 2 % Ammonium acetate: Isopropyl alcohol: Triethyl -amine (4:6:0.1 %v/v/v)

Wavelength: 242nm

Linearity range: 125 -750 ng/band EMPA; 5000 –30000 ng/band MET

%Recovery: 99.20 –101.50 % EMPA; 99.05 -102.54 % MET

LOD: 24.65ng/band EMPA; 705.21ng/band MET

LOQ: 74.70ng/band EMPA; 2136.99ng/band MET

18

4

HPTLC

Stationary Phase: Silica gel 60 F254

Mobile Phase: Toulene: 3%Ammonium acetate in methanol: ethyl- acetate: Ammonia (3:5:2:0.4 %v/v/v)

Wavelength: 230 nm

Linearity range: 500-2500 ng/band EMPA; 500-2500 ng/band MET

%Recovery: 99.89–101.91 % EMPA; 98.56 -101.80 % MET

LOD: 38.03 ng/band EMPA; 41.86 ng/band MET

LOQ: 115.24 ng/band EMPA; 126.85 ng/band MET

19

5

UV

Wavelength: 224nm EMPA; 230nm MET

Solvent: Methanol

Linearity range: 1-3µg/ml EMPA; 10-50µg/ml MET

LOD: 0.036µg/ml EMPA; 0.04µg/ml MET

LOQ: 0.111 µg/ml EMPA; 0.1402 µg/ml MET

20

 

Table – 4: Methods for Empagliflozin and Metformin

S. No.

Drugs and Method

Description

Ref. No.

1

EMPA

(RP-HPLC)

Stationary Phase: C18 column (100 mm X2.5 mm; 5 µm)

Mobile Phase: Methanol: Water with 0.1 % Ortho phosphoric acid (70:30 % v/v)

Wavelength: 296 nm

Flow rate: 0.8ml/min

Linearity range: 2.5–1.50 µg/ml

Retention time: 1.283 min

21

2

EMPA

(RP-HPLC)

Stationary Phase: Phenomenex C18 column (250 mm X4.6 mm; 5 µm)

Mobile Phase: Methanol: Water (70:30 % v/v)

Flow rate: 1 ml/min

Linearity range: 2–14 µg/ml

22

3

EMPA

(UV)

Wavelength: 224 nm

Solvent: Methanol: Water (9:1)

Linearity range: 1-3 µg/ml

LOD: 0.036 µg/ml

LOQ: 0.111 µg/ml

23

4

EMPA

(UV)

Wavelength: 223 nm

Solvent: Methanol and Water

Linearity range: 1-30 µg/ml

LOD: 0.10 µg/ml

LOQ: 0.33 µg/ml

24

5

MET

(UV)

Wavelength: 228-236 nm

Solvent: Distilled water

Linearity range: 1-14 µg/ml

LOD: 0.365 µg/ml

LOQ: 1.109 µg/ml

25

6

GEM and MET

(RP-HPLC)

Stationary Phase: Zorbax eclipse XDB C18 column (250 mm X4.6 mm; 5 µm)

Mobile Phase: Methanol: Water (20:80 % v/v)

Wavelength: 233 nm

Flow rate: 1 ml/min

Linearity range: 1–35 µg/ml GEM; 2-18 µg/ml MET

Retention time: 2.3 min GEM; 2.18 min MET

26

7

MET and DAPA

(RP-HPLC)

Stationary Phase: Cosmosil C18 column (250 mm X4.6 mm; 5 µm)

Mobile Phase: Methanol: Potassium dihydrogen Ortho phosphate buffer

(80:20 %v/v)

Wavelength: 228 nm

Linearity range: 100-500 µg/ml MET; 1-5 µg/ml DAPA

Retention time: 3.6 min MET; 5.2 min DAPA

LOD: 0.837 µg/ml MET; 0.052 µg/ml DAPA

LOQ: 0.158 µg/ml MET; 2.538 µg/ml DAPA

27

8

MET and REPA

(RP-HPLC)

Stationary Phase: Hypersil ODS C18 column (250 mm X4.6 mm; 5 µm)

Mobile Phase: Acetonitrile: 0.05 M Ammonium Acetate (60:40 %v/v)

Wavelength: 271 nm

Linearity range: 200-1200 µg/ml MET; 0.5-3 µg/ml REPA

Retention time: 3.13 min MET; 10.01 min REPA

LOD: 0.00037 µg/ml MET; 0.00016 µg/ml REPA

LOQ: 0.0012 µg/ml MET; 0.00048 µg/ml REPA

28

9

MET and PIO

(RP-HPLC)

Stationary Phase: BDS Hypersil C18 column (250 mm X4.6 mm; 5 µm)

Mobile Phase: Acetonitrile: Potassium dihydrogen ortho phosphate (50:50 %v/v)

Wavelength: 238 nm

Linearity range: 40-240 µg/ml MET; 12-72µg/ml PIO

Retention time: 2.81 min MET; 4.57 min PIO

29

*GEM-Gemigliptin, DAPA-Dapagliflozin, REPA-Repaglinide, PIO-Pioglitazone

 

CONCLUSION:

The above study presents analytical methods for the estimation of Empagliflozin, Linagliptin and Metformin hydrochloride in bulk materials and pharmaceutical dosage forms. Literature survey suggested that various RP-HPLC, HPTLC, TLC, UV methods were developed and reported. Hence all these methods are used to estimate and validate these in simple, economic, precise, accurate and reproducible ways. This review suggested that liquid chromatographic methods are widely used for estimation of drugs like Empagliflozin, Linagliptin and Metformin Hydrochloride. This review will help in future to develop the novel analytical methods for the approved antidiabetic drugs.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Vignan Pharmacy College, Vadlamudi, for providing all necessary facilities to carryout this review work.

 

CONFLICTS OF INTEREST STATEMENT:

All the authors declare that they do not have any conflicts of interest.

 

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Received on 13.05.2022       Modified on 23.10.2022

Accepted on 06.12.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2023; 13(1):42-46.

DOI: 10.52711/2231-5675.2023.00007