Analytical Methods of Antidiabetic Drugs –

Sitagliptin, Saxagliptin, Linagliptin, Alogliptin, Gemifibrozil, Troglitazone, Pioglitazone and Rosiglitazone: A Review

Varma. D. S.*, Dighe P. R.

S.M.B.T. College of Pharmacy, Dhamangaon, Tal. Igatpuri, Dist. Nashik.

*Corresponding Author E-mail: darshanavarma21019@gmail.com, darshavarma1999@gmail.com

ABSTRACT:

Analytical techniques used to determine chemical or physical property of analyte, chemical substance, chemical element or mixture. There are different types of analytical techniques used in pharmaceutical field for qualitative and quantitative estimation of drugs in biological fluids such as human plasma, human serum, human urine etc. and its formulations such as tablet, capsule, bulk drugs etc. This review work aims to provide most of existing analytical methods for analysis of Dipeptidyl peptidase-4 (DPP4) inhibitor and Peroxisome proliferator activated (PPAR) agonist. Estimation of Sitagliptin, Saxagliptin, Alogliptin, Gemifibrozil, Troglitazone, Pioglitazone, Linagliptin, and Rosiglitazone in active pharmaceutical ingredients, biological fluids and in various formulations which is reported in various research articles are reviewed. The analytical techniques which are utilised for the estimation of these drugs involves RP-HPLC, HPTLC, UPLC-MS/MS, LC-MS/MS, MALDI-TOF mass spectrometry, Raman spectroscopy, Liquid chromatography and Tandem mass spectrometry, TLC-densitometry with fluorescent detector, LC-ESI-MS-MS etc. are involved. The review assist in appropriate selection of analytical technique, mobile phase, column and detector based on available analytical instruments and chemicals by referring tabulated extensive condition. DPP4 inhibitor used in treatment of type 2 diabetes, hypoglycemia, weight loss etc. PPAR agonist also used in treatement of type 2 diabetes, in metabolic syndrome mainly for lowering triglycerides and blood sugar etc.

KEYWORDS: Analytical techniques, Biological fluids, DPP4 inhibitor, PPAR agonist, Diabetes.

INTRODUCTION:

Analytical technique may be defined as an instrument which is exciting and fascinating part of chemical analysis that interact with all the areas of chemistry and with many other field of pure and applied science. Analytical instrumentation play an important role in production and evaluation of new products.

The instrument provides the lower detection limits required to assure safe food, drug, water and air. (1) The analytical technique mainly based on four main principles these are electrochemical, spectroscopy, chromatographic and hyphenated techniques. (2,3) Diabetic mellitus is chronic metabolic disorder which is characterized by hyperglycemia and associated with impaired fat, protein and carbohydrates metabolism. (4) Diabetes cause impairment and eventually often kills, in large measure, because of the secondary effect of excessively elevated circulating and tissue glucose level. (5) For this review article drugs belong to DPP4 inhibitor and PPAR agonist are seleted. These drugs are Sitagliptin, Saxagliptin, Linagliptin, Alogliptin, Gemifibrozil, Troglitazone, Pioglitazone and Rosiglitazone

Review on Analytical Methods of Antidiabetic Drugs:

A. Sitagliptin

Molecular formula - C₁₆H₁₅F₆N₅.

IUPAC name - (3R)-3amino-1-[3-(trifluoromethyl)-5,6 dihydro [1,2,4] triazolo[4,3-al] pyrazine- 7(8H)-yl]-4-(2,4,5 trifluoro phenyl) butanol one phosphate.

It belongs to category Dipeptidyl peptidase 4 inhibitors (DPP4). The Sitagliptin was 1st DPP4 inhibitor drug authorized in year 2006. DPP4 inhibitor are novel agent for treatment of type 2 diabetes. Sitagliptin inhibits the enzyme DPP-4, in order to prevent the inactivation of GIP (Gastric inhibitory polypeptide) and GLP-1 (Glucagon-like peptide-1), thereby increasing insulin secretion from β-cells and decreasing glucagon release from the pancreas to improve the glycemic control in blood.

Table no 1: - Analytical methods of Sitagliptin

Technique	Extensive Method Condition	Detection outline	Form	Reference
LC-MS/MS	Column: - Fast LC C18 - 100 × 2.1mm ID; 2.7µm UHPLC EC C18 - 5×2.1nm ID; 2.7µm Mobile phase - 0.1% formic acid and methanol (45:55v/v). Flow rate -0.45ml/min at 30ºC Filter - Nylon membrane (0.2µm,25mm) Polytetrafluoroethylene membrane (0.20µm; 25mm filter).	_	tablet and human plasma	6
MALDI –TOF mass spectrometry	Solvent - Tertbutyl methyl ether: dichloromethane (80:20 v/v) Volume plasma - 10µl. Subjected analyte - 100W microwave Irradiation. Limit quantitation and detection -0.03µg/ml and 0.01µg/ml. Radiation time - ∼120s	_	API	7
Raman Spectroscopy	Illuminated -10× objective 30s Detector -Air cooled charged coupled 15 Raman Spectrum -200 to1800 cm ^-1wavenumber range. Spectral resolution - ∼9cm^-1	785nm laser	Solid dosage formulation	8
HPLC	Mobile phase: -n-heptane; ethanol: dimethylamine (35:65:0.1v/v/v). Stationary phase -Column: -AD-H (250nm×4.6mm, 5µm). Flow rate- 1.0ml/min. Limit detection and quantification -150 and 400ng/ml.	λ - 265nm	API	9

Table no 2: - Analytical methods of Saxagliptin

Technique	Extensive Method Condition	Detection outline	Form	Reference
Liquid Chromatography and Tandem mass Spectrometry	Column C18  - 50mm×2.1nm, 5µm Dynamic range  - 0.1-50ng/ml. Mobile phase  - methanol: acetonitrile: formic acid (50:50:0.1%v/v/v). Retention time -2.3min Mode - Positive ionization	_	API	10
RP-HPLC	RP-HPLC Method - Column C18 -4.6×150nm, 2.6µm. Mobile phase - acetonitrile: phosphate. (13:87v/v). Mode - Isocratic elution Flow rate - 1.5ml.min. Spector-densitometric method - Mobile phase - chloroform: methanol: formic acid. (80:20:0.3 v/v/v)	UV - λ -220nm UV -  λ -210nm	API	11
RP-HPLC	Colum C18 - 4.6×250nm, 5µm. Mobile phase - ortho phosphoric acid: methanol: acetonitrile, (70:10:20 v/v/v) Buffer: -Potassium dihydrogen phosphate adjusted pH:-4 Flow rate -1ml/min Mode  - Isocratic	λ -215 nm DAD detector	Pharmaceutical dosage	12
HPTLC	Mobile phase - hexane: methanol: ethyl acetate (4:2:4v/v/v) Retention factor - 0.18 Correlation coefficient: - 0.9995 Performed -Silica gel 60-F254, Aluminium plate	λ -210nm	API	13

B. Saxagliptin: -

Molecular formula: - C₁₈H₂₅N₃O₂.

IUPAC name :-(1s,3s,5s)-2-[(2s)-2-amino-2-(3-hydroxy-1-adamantyl) acetyl]-2-azabicyclo [3.1.0] hexane-3-Carbonitrile

Saxagliptin is class of DPP4 inhibitor class. It is used as oral hypoglycemic drug. Saxagliptin used in combination or as monotherapy with other drug treatment of type 2 diabetes. Its function to inhibit protein/enzyme and result in enhancing amount of active incretin. GIP and GLP-1

C. Linagliptin: -

Molecular formula: - C₂₃H₂₆N₈O₂.

IUPAC name:-8-[(3R)-3-aminopiperidin-1-yl]-7-but-2-yl-3-methyl-1-[(4-methylquinazolin-2-yl) methyl] purine-2, 6-dione

Linagliptin is class of DPP4 inhibitor belonging to class Gliptin. Linagliptin increases work of beta- cell and Insulin secretion, steady gastric emptying and declining HlbAlc level.

Table no 3: - Analytical methods of Linagliptin

Technique	Extensive Method Condition	Detection outline	Form	Reference
LC-MS/MS	Column -50×2.1mm, 3.5µm Mobile phase - 2mM ammonium acetate buffer and acetonitrile. Detection - Electrospray ionization Mode - Positive ionization m/z 473.2-420.2	_	API	14
TLC-densitometry with Fluorescent detection	Separation performed -Aluminium plate percolated with silica gel 60 F254 Mobile phase -tolune: methanol: ethylacetate (4:3:2v/v/v) Mode -Scanned in reflectance or Fluorescence	λ - 228nm fluorescence Emission	Pharmaceutical formulation and human plasma	15

Table no 4: - Analytical methods of Alogliptin

Technique	Extensive Method Condition	Detection outline	Form	Reference
RP-HPLC	Column  - 250mm, 4.6mm, 5µ Mobile phase  -acetonitrile: phosphate buffer: 0.3% sodium heptane sulfonate in water (60:20:20v/v/v) Phosphate buffer - 0.01M adjusted to pH 2.5 with O-phosphoric acid. Flow rate -1mlminˉ1	UV  - λ -220nm	Tablet	16
Spectrophotometric	Bromate-Bromide Mixture - Method A - Measured - Methylene orange Detection limit -0.115µgmlˉ1 Method B - Measured - methylene blue Detection limit - 0.210µg	λ -505nm λ -720nm	Bulk and Tablet	17
HPLC	Column - 250×4.6mm, 5µm Mobile phase - Solution A - water: acetylcholine: trifluoroacetic acid (1900:100:1v/v/v) Solution B - acetonitrile: water: trifluoroacetic acid (1900:100:1 v/v/v). Flow rate: - 1.0ml/min. Volume Injection - 20µl	UV detection -λ- 278nm	Bulk drug and tablet	18

D. Alogliptin:

Molecular formula: - C₁₇H₂₃N₅O₂.

IUPAC name: - 2-[(6-[(3R)-3-aminopiperdine-1-yl]-3-methyl-2, 4-dioxopyrimidin-1-yl] benzo nitrile

Alogliptin belong to class DPP4 inhibitor and used in therapy of type 2 diabetes mellitus. It used as Oral Hypoglycemic drug.

E. Gemfibrozil: -

Molecular formula: - C₁₄H₂₂O₃.

IUPAC name: - 5-(2, 5 dimethyl phenoxy)-2, 2-dimethylpentanoic acid.

Gemfibrozil is fibric acid derivatives used in therapy of dyslipidemia. Gemfibrozil result in various drug metabolism including statins by inhibiting sinusoidal influx transporter OATPIBI and also inhibit CYP2C8 and CYP2C9 enzyme.

Table no 5: - Analytical methods of Gemfibrozil

Technique	Extensive Method Condition	Detection outline	Form	Reference
UPLC-MS/MS	Column C18 -2.1×50mm., 1.7µm Mobile phase - 0.01% formic acid : acetonitrile (40:60v/v) Flow rate - 0.4ml/min Mode - Negative ionization m/z 249.2>121.1	_	API in plasma	19
Spectrofluorometric	Method determination- 0.10-6µgmlˉ1 aqs solution Detection and quantification limit - 0.03 and 0.01µgmlˉ1 Lower detection limit - 0.02µgmlˉ1	Excitation -λ-276nm Emission - λ-304nm	Pharmaceutical preparation	20
RP-LC	Column - 250×4.0mm I.D, 5µm Mobile phase - 0.4% phosphoric acid: acetonitrile (45:55 v/v) Detection limit - 0.025µgmlˉ1	Fluorescence detection	Human plasma	21

Table no 6: -Analytical methods of Troglitazone

Technique	Extensive Method Condition	Detection outline	Form	Reference
Semi- micro HPLC	Semi micro OJ-RH Column - 1.0mm I.D× 150mm, 5µm Mobile phase - methanol: acetic acid (1000:1 v/v). Flow rate - 20µl/min Volume Injection - 5µl	Electrochemical	Stereoisomer	22
HPLC potency assay	Column -120A (4.6×150mm, 5µm) Mobile phase - Isocratic elution - 50mM aqs NaH2PO4 at pH 4.0: acetonitrile: methanol (35:50:15 v/v/v) Flow rate - 1.0ml/min Injection volume - 10µl	UV - λ- 225nm	Tablet	23

F. Troglitazone: -

Molecular formula: - C₂₄H₂₇NO₅S.

IUPAC name:-(±-5-[(4-[3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran -2-yl) methoxy)phenyl)methyl]2,4-thiazolidinedione).

Troglitazone belong to new class knows Thiazolidinedione’s. Troglitazone work by reducing insulin resistance and decreases blood glucose by enhancing target cell response to insulin in muscle and adipose tissue and inhibit hepatic gluconeogenesis.

G. Pioglitazone: -

Molecular formula: - C₁₉H₂₀N₂O₃S.

IUPAC name: - (3-ethyl-4-methyl-N-{2-[4-({[(4-methylcyclohexyl) Carbamoyl] amino} sulfonyl) phenyl) ethyl}-2, 5-dihydro-1H-pyrrole-1-Carboxamide.

Pioglitazone belong to class Thiazolidinediones. The Thiazolidinediones are peroxisome proliferator activated receptor gamma (PPARγ) agonist and strike insulin resistance directly. Pioglitazone decreases risk of secondary macro-vascular trial and primary endpoint in patient with type2 Diabetic.

Table no 7: - Analytical methods of Pioglitazone

Technique	Extensive Method Condition	Detection outline	Form	Reference
HPLC	Double beam UV Visible spectrometry RP C18 - 25×0.46cm, 5µm Mobile phase - methanol: water (65:35v/v) Flow rate - 1ml minˉ1 Detection limit - Isosbestic Diluent - methanol: water (70:30v/v)	UV spectrophotometry λ- 230nm	Tablet	24
Liquid chromatography and Tandem mass Spectrometry	Column C18 - 100×4.6mm, 5µm Binary mobile phase - methanol: 5mM ammonium acetate in 0.1% formic acid (80:20v/v) Flow rate -  1ml/min Sample Injection - 10µl Runtime - 2.5min Mode - Positive ionization	_	Human plasma	25
Liquid chromatography and Tandem mass Spectrometry (Simultaneous determination of Pioglitazone and Glimepiride)	Column C18 - 4.6mm×100mm, 3.5µm Mobile phase - methanol: water: 5mM ammonium acetate in formic acid. (95:5:0.1v/v/v). Flow rate - 0.8ml/min Elution time - 2.4min m/z 496.2 -357.2	_	Tablet	26

Table no 8: - Analytical methods of Rosiglitazone

Technique	Extensive Method Condition	Detection Outline	Form	Reference
LC-MS/ MS	Column C18 - 50mm×2.1mm, 1.3µm Mobile phase - 0.1% formic acid in water or methanol Flow rate - 0.25ml/min Volume Injection:- 1µl Mode - Positive ionization Retention time - 1.6 min	_	API	27
HPLC	Column C18 - 250mm×4.6mm, 5µm Mobile phase - potassium dihydrogenphospate (PH-7): acetonitrile (60:40). Flow rate - 1.0ml/min Retention time - 17.36 min	UV Visible λ -225nm	API	28
LC-ESI-MS-MS	Column - 2.1mm×50mm, 5µm Mobile phase - 40%A and 60%B Mobile phase A - 10mM ammonium acetate with 0 02% trifluoroacetic acid Mobile phase B - 10mM ammonium acetate with acetonitrile Flow rate - 0.3 ml/min Volume injection  - 25.0µl Mode:- Positive ionization Retention time - ⁓1.8min Detected retention time - 1.05min m/z 358.1 and 362.1	_	Drug in human plasma	29
HPLC	Column C18 - 110°A Mobile phase - acetonitrile : 10mM ammonium acetate buffer (PH 5.2)  (43.5: 56.5 v/v) Flow rate -1ml/min Plasma calibration curve linear range - 2.5-250ng/ml	Excitation -λ- 247nm Emission - λ-367nm	Sheep plasma	30
Liquid chromatography/ Tandem mass spectrometry	Column C18 -100mm×2.0mm Particle size -3µm Mobile phase - acetonitrile: 0.1 %formic acid (60:40v/v) Detection and quantification - mass spectrometry Mode - Positive ionization	_	API in human plasma	31

Rosiglitazone: -

Molecular formula: - C₁₈H₁₉N₃O₃S.

IUPAC name:- 5-[(4-[2-(methyl-pyridin-2-ylamino) ethoxylphenyl) methyl]-thiazolidine-2-4-Dione. Rosiglitazone is synthetic PPARγ activator increasing PAH by inhibiting NOX4 expression and signaling reactive oxygen species generation and pulmonary arterial cell proliferation. The activation of PPARγ receptor cause pulmonary arterial vasodilation and reverses the pathological changes occur in PAH.

CONCLUSION:

Dipeptidyl peptidase 4 inhibitor and Peroxisome proliferator activated receptor agonist are used for the treatment of Diabetes. Various analytical methods tabulated for Sitagliptin, Saxagliptin, Linagliptin, Alogliptin, Gemfibrozil, Troglitazone, Pioglitazone and Rosiglitazone were obtained from literature survey of references book and research articles. The developed method supported in understanding the Pharmacokinetics, Pharmacodynamics, therapeutic drug monitoring, drug interactions with the organism, developing pharmaceutical formulation and determining the toxicity of these compounds. The routine analysis also assist the quality of formulation and give idea about further improvement of technique. This survey involves simple spectrophotometric technique as well as novel analytical technique such as HPLC, HPTLC, GC, LC-MS, GC-MS. Tabulated extensive method condition support analyst in selection of analytical technique, mobile phase, deter of drug, solvent and column.

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Received on 31.05.2022         Modified on 12.08.2022

Accepted on 22.09.2022   ©AandV Publications All Right Reserved

Res.  J. Pharma. Dosage Forms and Tech.2022; 14(4):324-330.

DOI: 10.52711/0975-4377.2022.00053