INTRODUCTION:

Linagliptin is an oral drug that reduces blood sugar (glucose) levels in patients with type 2 diabetes.¹ Linagliptin is a member of a class of drugs that inhibit the enzyme, dipeptidyl peptidase-4 (DPP-4). Other members of this class include saxagliptin (Onglyza), and sitagliptin (Januvia). After a meal, incretin hormones such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are released from the intestine, and their levels increase in the blood. GLP-1 and GIP lower down blood glucose level by increasing the production and release of insulin from the pancreas. GLP-1 also lower down blood glucose level by reducing the secretion by the pancreas of the hormone, glucagon, a hormone that increases the production of glucose by the liver and increase the blood level of glucose. The net effect of increased release of GLP-1 and GIP is to decrease blood glucose levels. Linagliptin inhibits the enzyme, DPP-4, that break down GLP-1 and GIP and thereby increases the levels and activity of both hormones. As a result, levels of GLP-1 and GIP in the blood remain higher, and blood glucose levels fall.² In summary, Linagliptin reduces blood glucose levels by inhibiting DPP-4 and increasing the levels of GLP-1 and GIP. Linagliptin was approved by the FDA in May 2011.^3,4

Fig.1. Structure of linagliptin.

Linagliptin may be taken with or without food. The recommended dose is 5mg/day. The most Common side effects of Linagliptin are stuffy/runny nose and sore throat. Hypoglycemia (low blood sugar level) may occur when Linagliptin is combined with insulin or a sulfonylurea-type drug. Some allergic reactions and muscle pain also may occur. Pancreatitis also has been reported. Rifampin lowers down the blood concentration of Linagliptin by stimulating break down of Linagliptin by CYP3A4 liver enzymes. Other drugs that increase activity CYP3A4 may also decrease the blood concentration of Linagliptin. Very few methods have been developed for the estimation of Linagliptin in pharmaceutical dosage forms by HPLC.⁵ Various techniques have been reported in the determination of linagliptin in the pharmaceutical dosage forms. The objective of review article is to understand the various simple accurate RP-HPLC methods with short retention time (migration time) and various methods to validate the newly developed method as per the ICH guidelines. In the present review; we have compiled the published analytical methods reported so far in the determination of linagliptin in pharmaceutical and biological samples. Techniques like spectrophotometry, HPLC, UPLC, LCMS, LC-ESI-MS where have been used for analysis, from which HPLC methods were been extensively adopted.⁶

MECHANISM OF ACTION:

Linagliptin is a dipeptidyl peptidase-4 inhibitor developed by Boehringer Ingelheim (German Pharmaceutical Company) for treatment of type II diabetes⁷. It was approved by the US FDA on 2 May 2011 for treatment of type II diabetes. DPP-4(dipeptidyl peptidase 4) is an enzyme that breaks down the incretion hormones, glucagon like peptide-1 (GLP-1) and glucose- dependent insulinotropic polypeptide like (GIP). These two GLP-1 and GIP increase insulin biosynthesis and secretion from pancreatic beta cells in the presence of normal and higher blood glucose levels. GLP-1 also lower down glucagon secretion from pancreatic alpha cells, resulting in a reduction in hepatic glucose output. Thus, linagliptin stimulates the release of insulin in a glucose dependent manner and decreases the levels of glucagon in the circulation, and showed that the drug can effectively reduce blood sugar.⁷

The Inhibitors of dipeptidyl peptidase 4 (DPP-4 inhibitors or gliptins) are a class of oral hypoglycemics agent that block the enzyme dipeptidyl peptidase-4 (DPP-4). They can be used to treat diabetes mellitus type 2. The first agent of the class – sitagliptin – was approved by the FDA in 2006.⁸ Glucagon increases blood glucose levels, and DPP-4 inhibitors reduce glucagon and thus blood glucose levels. The mechanism of DPP-4 inhibitors is to increase incretin levels (GLP-1 and GIP).⁹ which inhibit glucagon release, which in turn increases insulin secretion, decreases gastric emptying, and decreases blood glucose levels. A 2018 meta-analysis found no favorable effect of DPP-4 inhibitors on all-cause mortality, cardiovascular mortality, myocardial infarction or stroke in patients with type 2 diabetes.¹⁰

Fig.2.Mechanism of action of Linagliptine

Pharmacodynamics / Pharmacokinetics:

Preclinical, phase 1 and phase 2 studies provide evidence as to the pharmacokinetic and pharmacodynamics properties of linagliptin. Both in vitro and in vivo analyses showed linagliptin to be a potent and competitive DPP-4 inhibitor, with 50% of the DPP-4 enzyme inhibited at a mean concentration of approximately1 nM. In comparison, the concentration required to inhibit DPP-4 by 50% was 19nM and 50nM for sitagliptin and saxagliptin, respectively. The strong binding affinity of linagliptin and DPP-4 enzyme complex is further demonstrated by dissociation kinetics of around 200 hours, irrespective of dose. In addition to potency, linagliptin retains >10,000-fold selectivity for DPP-4 in relation to other dipeptidyl peptidases.¹¹ After multiple doses ranging from 2.5 to 10 mg, inhibition of DPP-4, 24 hours post dose, was sustained at 82% to 90%.¹² These properties allow for sustained inhibition of DPP-4, translating into the once-daily dosing of linagliptin. At the approved dose of 5 mg, the peak plasma concentration of 8.9nmol/L occurs at 1.5 hours post dose, yielding a mean plasma area under the curve of 139nmol·h/L. Linagliptin displays an absolute bioavailability of approximately 30%. Although high-fat meals reduce the peak plasma concentration by 15% and increase total exposure by 4%, this effect is not clinically significant. Linagliptin extensively distributes into tissues and displays concentration-dependent plasma protein binding. The percentage of the drug bound to protein decreases from 99% to approximately 82% as the plasma concentration increases from 1 nmol/L to ≥30 nmol/L. This reflects binding saturation at the DPP-4 enzyme and it is not altered in patients with renal or hepatic impairment. Another factor that is unaltered by renal or hepatic impairment is the effective half-life for accumulation of linagliptin after multiple oral doses (12 hours). About 90% of linagliptin is excreted unchanged and a small fraction is metabolized to an inactive metabolite. The majority of elimination (80%) occurs via the enter hepatic system, with a minor portion (5%) removed through the urine. Analysis of pharmacokinetics in patients with mild, moderate, and severe renal impairment demonstrated that dose adjustment is not required.¹³

Dosage/Administration/Availability:

Linagliptin is available in 5 mg tablets. The recommended dosage is 5mg once daily, administered with or without food. Dosage adjustments for renal or hepatic impairment are not necesssary. If linagliptin is used in combination with an insulin secretagogue, such as a sulfonylurea, the dosage of the secretagogue may require reduction to alleviate the risk of hypoglycemia. Currently, most private insurance companies are likely to list Tradjenta as a third tier drug or require proof of step therapy and physician authorization for approval. Most state Medicaid programs will also implement these restrictions.¹³

Overview of Analytical Method:

Analytical chemistry is the overall study of separation; identification and quantification of chemical components of natural and artificial materials incorporate with one or more compounds or elements. Analytical chemistry is divide into two main class based on analysis, qualitative analysis, that is to say the recognition with regard to the chemical components exist in the sample, whereas quantitative analysis measure the numerical amount or concentration of certain element or compound present in the substance i.e., sample.¹⁴ Pharmaceutical analysis plays a extremely impressive role in the examination of pharmaceutical formulations and bulk drugs with respect to the quality control and assurance. Large Number of drugs or compounds (New entities or partial structural modification of the existing one) enter in the market, speedy increase in pharmaceutical industries.¹⁵ development in analytical instruments and production of drug all over the world bring towards a increase in inevitable demand to seek novel and systematic analytical techniques in the pharmaceutical industries. The advancement of the analytical method development and analytical instruments have shorten the time and cost of analysis and enhanced precision and accuracy.¹⁶ the techniques use to analysis are developed and validated for API , drug products, excipients, related substances, degradation products and residual solvents etc. successively which become an essential part of the required necessities for regulatory organization.¹⁷ Analytical method development finally results in official test methods. In Consequence quality control laboratories used these methods to check the efficacy, identity, purity, safety as well as performance of products of the drug. This review focus on different analytical methods like Uv, HPLC, UPLC, LC-MS for the estimation of linagliptin in pharmaceutical formulations.¹⁸

Table 1: Summery of Analysis of Linagliptin by UV-Spectroscopy methods.

No.	Drug	Method	Description	Ref
1.	Assay of Linagliptine in Bulk and Marketed Dosage Form	UV- Spectroscopic Method	Detection wavelength: 294 nm in Methanol Linearity range: 5-30 µg/ml Co-relation Co-efficient: 0.999 % Recovery range: 99.43-100.01 % %RSD: ≤2%	19
2.	Determination of Linagliptine in Bulk and Pharmaceutical Dosage Forms	UV- Spectroscopic Method	Detection wavelength: 294 nm in Methanol Linearity range: 5-25 µg/ml Co-relation Co-efficient: 0.999 % Recovery range: 99.76-100.22 % %RSD: ≤2%	20
3.	Estimation of Metformin and Linagliptin	Stability Indicating UV- Spectroscopic Method	Detection wavelength: 294.4 nm for Linagliptin and 230.4 nm for Metformin Linearity range: 10-40 µg/ml for Linagliptin and 2-14 µg/ml for Metformin Co-relation Co-efficient: 0.999 for Metformin and 0.999 for Linagliptin % Recovery range: 96.66-100.75 % %RSD: ≤2%	21
4.	Estimation of Linagliptine	UV- Spectroscopic Method	Detection wavelength: 241 nm in Methanol and Water (50;50) Linearity range: 10-35 µg/ml Co-relation Co-efficient: 0.999 % Recovery range: 99.60-100.65 % %RSD: ≤2%	22
5.	Estimation of Empagliflozin and Linagliptin	UV- Spectroscopic Method	Detection wavelength: 277 nm for Linagliptin and 233 nm for Empagliflozin Linearity range: 2-6 µg/ml for Linagliptin and 5-15 µg/ml for Empagliflozin Co-relation Co-efficient: 0.999 for Empagliflozin and 0.999 for Linagliptin % Recovery range: 98-101 % %RSD: ≤2%	23
6	Estimation of Empagliflozin and Linagliptin	UV- Spectroscopic Method	Detection wavelength: 238 nm for Linagliptin and 221 nm for Empagliflozin Linearity range: 2.5-30 µg/ml for Linagliptin and 2.5-30 µg/ml for Empagliflozin Co-relation Co-efficient: 0.999 for Empagliflozin and 0.999 for Linagliptin % Recovery range: 99.8-100.3 % %RSD: ≤2%	24

Table 2: Summery of Analysis of Linagliptin by HPLC and UPLC technique.

No.	Drug	Method	Description	Ref
1.	Validated RP-HPLC method for the determination of linagliptin	RP-HPLC method	Column: Gemini C18 column (250x4.6mm, I.D, 5µm) Mobile phase: Methanol : Water (83:17v/v) Flow rate: 1mL/min Wavelength: 241nm Retention time: 5.85 min	25
2.	Spectrophotometric and Chromatographic Estimation of Linagliptin in Bulk and Tablet Dosage Form.	RP-HPLC method	Column: C8 (250 x 4.6 mm, 5µm) Mobile phase: Mixture of Phosphate buffer : Acetonitrile (35:65% v/v) Flow rate: 1mL/min Wavelength: 227nm Retention time: 2.41 min	26
3.	Quality by Design Based Hplc Assay Method Development and Validation of Linagliptin in Tablet Dosage Form.	RP-HPLC method	Column: C18 (150 x 4.6 mm, 5 µm) Mobile phase: Mixture of Phosphate buffer : Methanol (70:30 % v/v) Flow rate: 1mL/min Wavelength: 292nm Retention time: 7.2 min	27
4.	Stability Indicating HPLC-DAD Method for the Determination of Linagliptin in Tablet Dosage Form:	RP-HPLC method	Column: C18 (150 x 4.6 mm, 5µm) Mobile phase: Mixture of Methanol : Water (40:60 % v/v) Flow rate: 1mL/min Wavelength: 225nm Retention time: 11 min	28
5.	Method Development and Validation of Rp-Hplc Method for Determination of New Antidiabetic Agent Linagliptin in Bulk and in Pharmaceutical Formulation.	RP-HPLC method	Column: C18 (150 x 4.6 mm, 5µm) Mobile phase: Mixture of 0.02 M potassium dihydrogen phosphate : Acetonitrile (70:30% v/v) Flow rate: 1mL/min Wavelength: 226nm Retention time: 4.2 min	29
6.	A Rapid RP-HPLC Method development and Validation for the Analysis of Linagliptine Bulk and Pharmaceutical Dosage Form.	RP-HPLC method	Column: C18 (100 x 2.5 mm, 3µm) Mobile phase: Mixture of Methanol : Water (70:30% v/v) Flow rate: 0.8 mL/min Wavelength: 296nm Retention time:	30
7.	RP-HPLC method development and validation of Linagliptin in bulk drug and pharmaceutical dosage form.	RP-HPLC method	Column: C18 (100x4.6 mm, 5 µm) Mobile phase: Mixture of Phosphate buffer : Methanol (50:50 % v/v) Flow rate: 0.8 mL/min Wavelength: 238nm Retention time:	31
8.	Development and validation of stability indicating RP-HPLC method for simultaneous estimation of empagliflozin and linagliptin in tablet formulation	RP-HPLC method	Column: BDS C18 column (250X4.6mm ID, 5µm) Mobile phase: 0.1% perchloric acid : acetonitrile (60:40) Flow rate: 1mL/min Wavelength: 230 nm	32
9.	Simultaneous estimation of metformin and linagliptin in tablet dosage forms	RP-HPLC method	Column: Waters X- bridge C18 column (4.6X150mm ID, 5µm) Mobile phase: Acetonitrile e: 0.2M Phosphate Buffer (35:65 v/v) Flow rate: 1mL/min Wavelength: 225nm	33
9.	Simultaneous estimation and stability indicating study of Metformin and linagliptin in dosage forms	RP-HPLC method	Column: Inertsil ODS 3V C18 column (250X4.6mm ID, 5µm) Mobile phase: Phosphate buffer : acetonitrile (60:40) Flow rate: 1mL/min Wavelength : 280nm	34
10.	Development and validation of stability indicating RP-HPLC method for the simultaneous estimation of linagliptin and metformin	RP-HPLC method	Column: BDS Hypersil C8 column (250x4.6mm ID, 5µm) Mobile phase: Acetonitrile e : Water : Methanol (25:20:25 v/v) Flow rate: 1mL/min Wavelength : 243nm	35
11.	Determination of Metformin hydrochloride and linagliptin by RPHPLC in bulk and Pharmaceutical formulation	RP-HPLC method	Column: Phenomnex Luna RP-18 reverse phase C18 column (150x4.6mm ID, 5µm) Mobile phase: Phosphate buffer : Methanol : acetonitrile (65:10:25v/ v) Flow rate: 1mL/min Wavelength :231nm	36
12.	Stability indicating liquid chromatographic method for simultaneous assay of linagliptin and metformin pure and tablet dosage form	RP-HPLC method	Column: Zorbax CV18 column (250X4.6mm ID, 5µm) Mobile phase: Phosphate buffer : acetonitrile (40:60v/v) Flow rate: 1mL/min Wavelength: 236nm	37
13.	Simultaneous determination of linagliptin and metformin by RPHPLC method	RP-HPLC method	Column: Lichrosphere 100 RP -18e ( 125x4mm ID, 5µm) Mobile phase: Methanol: 0.05M potassium dihydrogen orthophosphate (70:30v/v) Flow rate: 1mL/min Wavelength : 267nm	38
14.	Development and Validation of ultra-performance liquid chromatography (UP-LC) method for estimation of a new anti-diabetic drug Linagliptin in bulk and its tablet formulation.	UPLC method	Column: SB-C18 (50 × 2.1 mm, 1.8 μm) Mobile phase: Mixture of Acetonitrile : 0.01M Potassium phosphate buffer (70:30% v/v) Flow rate: 0.3 mL/min Wavelength : 292nm	39

CONCLUSION:

There are a wide range of analytical techniques were available for the analysis of linagliptin in pharmaceutical and biological samples. HPLC with UV detection is applicable in the case of analysis of linagliptin in pharmaceuticals which provide us cost effective accurate method when compare with more advance techniques. This review depicts the reported Spectrophotometric and Chromatographic methods; developed and validated for estimation of Linagliptin alone or on combination with Metformin or Empagliflozin. According to this review it was concluded that for Linagliptin different Spectroscopic and Chromatographic methods are available for single component as well as for combination and also it was found that the mobile phase containing phosphate buffer, methanol and acetonitrile were common for most of the chromatographic method to provide more resolution. It was observed that most common combination of Linagliptin was with Metformin (ex. JENTADUETO, TRAJENTA). For chromatographic method flow rate is observed in the range of 0.6-1.2 ml/min to get good retention time. For number of the Spectroscopic methods common solvent is Methanol. Hence this all methods found to be simple, accurate, economic, precise, and reproducible in nature. From this elaborate literature review it was found that, till date there is no RP-HPLC method available for the determination of Linagliptin with Metformin or Empagliflozine using Design of Expert or Quality by Design.

ACKNOWLEDGMENT:

The authors are grateful to Pravara Rural College of Pharmacy Pravaranagar Loni, for providing necessary facilities.

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Received on 05.02.2021 Modified on 03.03.2021

Research Journal of Science and Technology. 2021; 13(2):127-132.

DOI: 10.52711/2349-2988.2021.00019