Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Linagliptin, Empagliflozin and Metformin in Solid Dosage Forms

T. Naga Ravi Kiran¹*, P. Parvathi², J.N. Suresh Kumar³

¹Faculty and Head, Department of Pharmaceutical Chemistry, Narasaraopeta Institute of Pharmaceutical Sciences, Kotappakonda Rd, Narasaraopeta, Andhra Pradesh 522601

²Research scholar, Department of Pharmaceutical Chemistry, Narasaraopeta Institute of Pharmaceutical Sciences, Kotappakonda Rd, Narasaraopeta, Andhra Pradesh 522601

³Principal, Narasaraopeta Institute of Pharmaceutical Sciences, Kotappakonda Rd, Narasaraopeta, Andhra Pradesh 522601

*Corresponding Author E-mail: ravikirannaga58@gmail.com

ABSTRACT:

The purpose of the investigation was to develop a simple, rapid and accurate RP-HPLC method to determine assay of Linagliptin, Empagliflozin and Metformin in solid dosage forms. The chromatographic separation was performed on Kromosil 250 x 4.6 mm, 5µm. Eluents were monitored on PDA detector at a wavelength of 233 nm using a Buffer: Acetonitrile (45:55v/v). The column temperature was maintained at 30°C. Validation parameters such as system suitability, linearity, precision, accuracy, specificity, limit of detection (LOD), limit of quantification (LOQ), Stability of sample and standard stock solutions and robustness were studied as reported in the ICH guidelines. The retention time for Linagliptin, Empagliflozin and Metformin was 2.370min, 2.787min and 3.419 min respectively. Assay method further evaluated for Linagliptin, Empagliflozin and Metformin analysis at low concentration of analyte and found limit of detection is 0.02 µg/ml, 0.08 µg/ml and 9.22µg/mlrespectively and limit of Quantitation is 0.05µg/ml, 0.25µg/mland 27.94µg/mlrespectively. The percentage recovery of Linagliptin, Empagliflozin and Metformin was 99.99%, 99.47% and 100.01% respectively. The %RSD for Linagliptin, Empagliflozin and Metformin was 0.5%, 0.7% and 0.2%respectively. Linearity of Linagliptin, Empagliflozin and Metformin performed from 25% to150% and the R² is 0.999, intercept and slope found to be y = 25605x + 1357, y = 40970x + 3932 and y = 1497.x + 12010 respectively. The method was fast, accurate, precise and sensitive hence it can be employed for routine quality control of Linagliptin, Empagliflozin and Metformin containing drug in quality control laboratories and pharmaceutical industries.

KEYWORDS: RP-HPLC, Linagliptin, Empagliflozin and Metformin.

INTRODUCTION:

Metformin Hydrochloride is a biguanide derivative which is the most commonly prescribed drug to treat hyperglycemia in individuals with Type 2 diabetes especially in overweight patients. Chemically it is known as1,1- dimethylbiguanide hydrochloride. Metformin decreases blood glucose levels by decreasing hepatic glucose production, decreasing intestinal absorption of glucose, and improving insulin sensitivity by increasing peripheral glucose uptake and utilization.(1) These effects are mediated by the initial activation of AMP-activated protein kinase (AMPK), a liver enzyme that plays an important role in insulin signaling, whole body energy balance, and the metabolism of glucose and fats. Empagliflozin is a sodium glucose cotransporter -2 (SGLT-2) inhibitor indicated as an adjunct to diet and exercise to improve glycemic control in adult patients with type-2 diabetes. Chemically it is known as (2S,3R,4R,5S,6R)-2-[4- chloro-3-[[4-[(3S)-oxolan-3- yl]oxyphenyl]methyl]phenyl]- (hydroxymethyl)oxane-3,4,5-triol. It is marketed under the trade name Jardiance. SGLT2 cotransporters are responsible for reabsorption of glucose from the glomerular filtrate in the kidney.(2) The glucuretic effect resulting from SGLT2 inhibition reduces renal absorption and lowers the renal threshold for glucose, therefore resulting in increased glucose excretion. Additionally, it contributes to reduced hyperglycaemia and also assists weight loss and blood pressure reduction. Linagliptin is chemically 8-[(3R)-3-aminopiperidin-1-yl]-7-(but-2-yn-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-2,3,6,7-tetrahydro-1H-purine-2,6-dione.Linagliptin belongs to the class of Dipeptidylpeptidase 4-inhibitor,protease inhibitor.It is verysoluble in Ethanol, Water. (3) The literature survey reveals that there are only few analytical methods available for estimation of Metformin Hydrochloride, Linagliptin and Empagliflozin individually and in combination are spectrophotometric and HPLC methods.(4-8)

So we have planned to develop a simple, precise, economic and accurate Stability indicating RP-HPLC method development and validation for the estimation of Linagliptin, Empagliflozin and Metformin in solid dosage forms.

MATERIALS AND METHODS:

Active pharmaceutical ingredients Metformin Hydrochloride, Linagliptin and Empagliflozin were obtained as a gift sample from Hetero Drugs Limited, Hyderabad. The solvents used in this work were of HPLC grade and obtained from Rankem.

Instrumentation and chromatographic conditions

The analysis was performed on a high-performance liquid chromatography system consists of waters 2695 with 2996 module Photo Diode Array detector equipped with a quaternary solvent delivery pump, automatic sample injector and column thermostat. The data acquisition and analysis was performed by using Empower2 software. The chromatographic separation was performed on Kromosil 250 x 4.6 mm, 5µm. The flow rate was kept at 0.8ml/min. The column temperature was maintained at 30°C. The mobile phase was made of 0.1% Ortho Phosphoric Acid Buffer and Acetonitrile taken in the ratio45:55 ratio had gave acceptable retention time and good resolution between Metformin Hydrochloride and Empagliflozin. The method was optimized at 233nm. Data acquisition and processing was performed by using empower system software. (9). The run time was taken as 6min. All the determinations are carried out at an ambient temperature.

Preparation of Standard stock solutions

Accurately Weighed and transferred 125mg of Metformin Hydrochloride and 12.5mg of Empagliflozin working Standards into a 25ml and 100 ml clean dry volumetric flasks respectively, add 3/4th volume of diluents (Water: Acetonitrile in the ratio 50:50), sonicated for 10minutes and make up to the final volume with diluents. 1ml from the above two stock solutions was taken into a 10ml volumetric flask and made up to 10ml with diluent. (10)

Preparation of Sample stock solutions

Accurately Weighed and transferred equivalent to 125mg of Metformin Hydrochloride and 12.5mg of Empagliflozin tablets powder into a 25ml and 100 ml clean dry volumetric flasks respectively, add 3/4th volume of diluent (Water: Acetonitrile in the ratio 50:50), sonicated for 15minutes and make up to the final volume with diluent and filtered. 1ml from the above two filtered stock solutions was taken into a 10ml volumetric flask and made up to 10ml with diluent. (11)

Preparation of buffer (0.1%OPA):

1ML of Ortho Phosphoric Acid solution in a 1000ml of volumetric flask add about 100ml of HPLC grade water and final volume make up to 1000 ml with HPLC grade water. (12)

Method validation: The method was validated according to ICH guidelines. The different validation characteristics which were performed are following: Linearity, accuracy, Precision, limit of detection, limit of quantification, robustness and the stability indicating capability.

System suitability parameters

The system suitability parameters were determined by preparing standard solutions of Metformin Hydrochloride, Linagliptin and Empagliflozin and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined. (13)

Precision:

Preparation of Standard working solutions (100% solution): 1ml from each standard stock solution was pipette out and taken into a 10ml volumetric flask and made up with Water: ACN (50:50 v/v). (1000ppm, 10ppm and 5ppm)

Preparation of Sample stock solutions: 5 tablets were weighed and calculate the average weight of each tablet then the weight equivalent to 1 tablet was transferred into a 100 mL volumetric flask, 25mL of diluent added and sonicated for 50 min, further the volume made up with diluent and filtered.

Preparation of Sample working solutions (100% solution): 1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluents. (1000ppm, 10ppm and 5ppm)

Linearity:

Preparation of Standard stock solutions: Accurately weighed 5mg of Linagliptin., 10mg of Empagliflozin and 1000mg of Metformin and transferred to three 25ml volumetric flasks separately. 10ml of diluent was added to flasks and sonicated for 15mins. Flasks were made up with Water: ACN (50:50 v/v) and labelled as Standard stock solution 1, 2 and 3.25% Standard solution: 0.25ml each from three standard stock solutions was pipette out and made up to 10ml. 50% Standard solution: 0.5ml each from three standard stock solutions was pipette out and made up to 10ml.75% Standard solution: 0.75ml each from three standard stock solutions was pipette out and made up to 10ml. 100% Standard solution: 1.0ml each from three standard stock solutions was pipette out and made up to 10ml. 125% Standard solution: 1.25ml each from three standard stock solutions was pipette out and made up to 10ml. 150% Standard solution: 1.5ml each from three standard stock solutions was pipette out and made up to 10ml. (14)

Accuracy:

Preparation of 50% Spiked Solution: 0.5ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Preparation of 100% Spiked Solution: 1.0ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Preparation of 150% Spiked Solution: 1.5ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.

Robustness conditions like Flow minus (0.9ml/min), Flow plus (1.1ml/min), mobile phase minus (55:45v/v), mobile phase plus (45:55v/v), temperature minus (25°C) and temperature plus(35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.(15)

LOD sample Preparation: 0.25ml each from three standard stock solutions was pipette out and transferred to 3 separate 10ml volumetric flask and made up with diluents from the above solutions 0.1ml, 0.1ml and 0.1ml of Linagliptin, Empagliflozin and Metformin solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluents.

LOQ sample Preparation: 0.25ml each from three standard stock solutions was pipette out and transferred to 3 separate 10ml volumetric flask and made up with diluents from the above solutions 0.3ml, 0.3ml and 0.3ml of Linagliptin, Empagliflozin and Metformin solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluents.(16)

Degradation studies:

Oxidation:

To 1 ml of stock solutions of Linagliptin, Empagliflozin and Metformin. 1 ml of 20% hydrogen peroxide (H₂O₂) was added separately. Thesolutionswere kept for 30 min at 60⁰C. For HPLC study, the resultant solution was diluted to obtain 5µg/ml, 10µg/ml and 1000µg/ml of all components and 10µlwereinjectedintothe system and the chromatograms were recorded to assess the stability of sample.(17)

Acid Degradation Studies:

To 1 ml of stock solution Linagliptin, Empagliflozin and Metformin, 1ml of 2N Hydrochloricacidwasadded and refluxed for 30mins at 60⁰C.The resultant solution was diluted to obtain 5µg/ml, 10µg/ml and 1000µg/ml of all components and10µl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.(18)

Alkali Degradation Studies:

To 1 ml of stock solution Linagliptin, Empagliflozin and Metformin, 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 60⁰C. The resultant solution was diluted to obtain 5µg/ml, 10µg/ml and 1000µg/ml of all componentsand 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.(19)

Dry Heat Degradation Studies:

The standard drug solution was placed in oven at 105⁰c for1h to study dry heat degradation. For HPLC study, the resultant solution was diluted obtain 5µg/ml, 10µg/ml and 1000µg/ml of all components and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.(19)

Photo Stability studies:

The photochemical stability of the drug was also studied by exposing the 50µg/ml, 100µg/ml and 10000µg/ml solution to UV Light by keeping the beaker in UV Chamber for 1days or 200-Watt hours/m²in photo stability chamber^.For HPLC study, the resultant solution was diluted to obtain 5µg/ml, 10µg/ml and 1000µg/ml of all componentsand 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.(20)

Neutral Degradation Studies:

Stress testing under neutral conditions was studied by refluxingthedruginwaterfor6hrs atatemperature of 60ºC. For HPLC study, the resultant solution was diluted to obtain 5µg/ml, 10µg/ml and 1000µg/ml of all components and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.(20)

RESULTS AND DISCUSSIONS:

Development and optimization of HPLC method:

The present work was focused to develop stability indicating RP-HPLC method for the simultaneous estimation of Metformin Hydrochloride, Linagliptin and Empagliflozin in solid dosage forms. The solubility of the active pharmaceutical ingredients were checked in different solvents like methanol, water, Acetonitrile and in different ratios but finally the standards were soluble in water: acetonitrile (50:50) so it was chosen as a diluent. The different mobile phases like Acetonitrile and potassium dihydrogen phosphate buffer and water: methanol were used in compositions with a flow rate of 1ml/min but the peak resolution, retention time and tailing factor were not satisfactory so at last 0.1% orthophosphoric acid and Acetonitrile was selected as a mobile phase at flow rate of 0.8ml/min. Initially “Hypersil BDS” (250mm x 4.6mm x 5µ) columns with different temperatures like 30, 35, 40, 45°C were used but the retention time, run time and peak resolution were not exact and the problem was get rid by using “kromosil18”(250mm x 4.6mm x 5µ) kept at 30°c with a run time of 6 minutes. Finally the method was optimized by altering the mobile phase composition / ratio and the optimized wavelength of three drugs Linagliptin, Empagliflozin and Metformin was found to be at 233nm.

Forced degradation studies:

The stability studies were conducted by exposing the dosage forms to different stress conditions like acid, base, peroxide, light and water. It was found that the dosage forms were slightly degraded in acid, base and peroxide but stable in photolytic and hydrolytic conditions.

System suitability parameters:

The system suitability tests were conducted before performing the validation and the parameters were within the acceptance criteria. Linagliptin, Empagliflozin and Metformin were eluted at 2.360 min, 2.763min and 3.265 min respectively with good resolution. Plate count and tailing factor was very satisfactory, so this method was optimized and to be validated. Drugs were eluted with good retention time, resolution; all the system suitable parameters like Plate count and Tailing factor were within the limits.

Plate count of the Linagliptin Was 3790±126, Empagliflozin was 4620±126, and of Metformin was 6406±126, tailing factor of Linagliptin was 1.26±0.5, Empagliflozin was 1.26±0.2, and of Metformin was 1.18±0.3, resolution between Linagliptin and Empagliflozin was 4.1 and resolution between Empagliflozin and Metformin was 3.1. According to ICH guidelines plate count should be more than 2000, tailing factor should be less than 2 and resolution must be more than 2. All the system suitable parameters were passed and were within the limits.

Linearity:

Six linear concentrations of Linagliptin (1.25-7.5µg/ml), Empagliflozin (2.5-15µg/ml) and Metformin (250-1500µg/ml) were injected in a triplicate manner. Average areas were mentioned above and linearity equations obtained for Linagliptin was y = 25605x + 1357. Empagliflozine was y = 40970x + 3932. and of Metformin was y = 1497.x + 12010. Correlation coefficient obtained was 0.999 for all the three drugs.

Precision:

System Precision

From a single volumetric flask of working standard solution six injections were given and the obtained areas were mentioned above. Average area, standard deviation and % RSD were calculated for three drugs and obtained as 0.5%, 0.7% and 0.2% respectively for Linagliptin, Empagliflozine and Metformin. As the limit of Precision was less than “2” the system precision was passed in this method.

Repeatability:

Multiple sampling from a sample stock solution was done and six working sample solutions of same concentrations were prepared, each injection from each working sample solution was given and obtained areas were mentioned in the above table. Average area, standard deviation and % RSD were calculated for three drugs and obtained as 0.5%, 0.5% and 0.2% respectively for Linagliptin, Empagliflozin and Metformin. As the limit of Precision was less than “2” the system precision was passed in this method.

Intermediate precision:

Multiple sampling from a sample stock solution was done and six working sample solutions of same concentrations were prepared, each injection from each working sample solution was given on the next day of the sample preparation and obtained areas were mentioned in the above table. Average area, standard deviation and % RSD were calculated for three drugs and obtained as 0.3%, 0.7% and 0.3% respectively for Linagliptin, Empagliflozin and Metformin. As the limit of Precision was less than “2” the system precision was passed in this method.

Accuracy:

Three levels of Accuracy sample were prepared by standard addition method. Triplicate injections were given for each level of accuracy and mean %Recovery was obtained as 99.99%, 99.77% and 100.01% for Linagliptin, Telmesartan and Metformin. Respectively.

Robustness:

Robustness conditions like Flow minus (0.9ml/min), Flow plus (1.1ml/min), mobile phase minus (55B:45A), mobile phase plus (55B:45A), temperature minus (25°C) and temperature plus(35°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.

Assay:

Trijardy XR (1000+10+5) the label claim Empagliflozin 10mgLinagliptin 5mg Metformin 1000mg per unit formulation Assay was performed with the above formulation. Average % Assay for Linagliptin, Empagliflozin and Metformin. Obtained was 99.63%, 100.05% and 100.12% respectively.

Degradation Studies:

Degradation studies were performed with the formulation and the degraded samples were injected. Assay of the injected samples was calculated and all the samples passed the limits of degradation

Conclusion

A simple, accurate, precise method was developed for the simultaneous estimation of the Linagliptin, Empagliflozin and Metformin in Tablet dosage form. Hence this method can be applied for the estimation of Metformin Hydrochloride and Empagliflozin in drug testing laboratories and pharmaceutical industries.

Table 1. System suitability parameters of Linagliptin, Empagliflozin and Metformin

Parameters		Linagliptin	Empagliflozin	Metformin	LIMIT
Linearity Range (µg/ml)		1.25-37.5µg/ml	2.5-15 µg/ml	250-1500µg/ml	R< 1
Regression coefficient		0.999	0.999	0.999
Slope(m)		25605	40970	1497
Intercept(c)		1357	3932.	12010
Regression equation (Y=mx+c)		y = 25605x + 1357	y = 40970x + 3932.	y = 1497.x + 12010
Assay (% mean assay)		99.63%	100.05%	100.12%	90-110%
Specificity		Specific	Specific	Specific	No interference of any peak
System precision %RSD		0.5	0.7	0.2	NMT 2.0%
Method precision %RSD		0.5	0.5	0.2	NMT 2.0%
Accuracy % recovery		99.99%	99.77%	100.01%	98-102%
LOD		0.02/ml	0.08 µg/ml	9.22µg/ml	NMT 3 µg/ml
LOQ		0.05µg/ml	0.25 µg/ml	27.94µg/ml	NMT 10µg/ml
Robustness	FM	0.6	0.5	0.4	% RSD NMT 2.0
	FP	0.4	0.2	0.3
	MM	0.3	0.2	0.2
	MP	0.2	0.9	0.9
	TM	0.3	1.0	0.2
	TP	0.3	0.7	0.2

Fig.1 Standard chromatogram of Linagliptin, Empagliflozin and Metformin

Fig 2. Calibration curve of Linagliptin

Fig 3. Calibration curve of Empagliflozin

Fig 4. Calibration curve of Metformin

Fig.5 Linearity chromatogram of Linagliptin, Empagliflozin and Metformin

Fig 6. System precision chromatogram of Linagliptin, Empagliflozin and Metformin

Fig 7. Repeatability chromatogram of Linagliptin, Empagliflozin and Metformin

Fig 8. Intermediate precision chromatogram of Linagliptin, Empagliflozin and Metformin

Fig 9. Accuracy 150% chromatogram of Linagliptin, Empagliflozin and Metformin

Fig 10. LOQ chromatogram of standard Linagliptin, Empagliflozin and Metformin

Fig 11. Acid Degradation chromatogram of Linagliptin, Empagliflozin and Metformin

Fig 12. Base Degradation chromatogram of Linagliptin, Empagliflozin and Metformin

Fig 13. Peroxide Degradation chromatogram of Linagliptin, Empagliflozin and Metformin

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Received on 27.07.2020 Modified on 02.08.2020

Asian J. Pharm. Ana. 2020; 10(3):117-124.

DOI: 10.5958/2231-5675.2020.00021.6