To develop and validate the stability indicating method for Empagliflozin and Linagliptin using HPLC method

 

Mohd Bilal Sufi; Vaishnavi Daldale*; Irshad Ahmed

 

Department of Pharmaceutical Quality Assurance, New Montfort Institute of Pharmacy, Ashti,

Dist – Wardha, (M.S) – 442202

 

*Correspondence: vaishnavidaldale27@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2026.5712         

Article Information		Abstract
Research Article Received: 14/07/2026 Accepted: 18/07/2026 Published:31/07/2026   Keywords Linagliptin; Empagliflozin; RP-HPLC; Method Validation; Forced Degradation.		The fixed-dose combination of linagliptin and empagliflozin for the treatment of type 2 diabetes mellitus is increasingly being used in clinical practice. Consequently, there is a need for stability studies and routine quality control testing of the drug product, which requires the development of a simple, reliable, and stability-indicating analytical method. This research was aimed to develop, optimize and validate the simultaneous determination of two compounds Linagliptin and Empagliflozin in their bulk and pharmaceutical tablet dosage form using reverse phase high-performance liquid chromatography (RP-HPLC) with ICH Q2(R2) criteria. The separation was performed by HPLC on a C18 column with mobile phase methanol/0.1% orthophosphoric acid (30:70 v/v) at 0.7mL/min flow rate at 235nm UV. The developed method was validated in terms of system suitability, linearity, accuracy, precision, repeatability, sensitivity, ruggedness, robustness, assay and forced degradation studies. The linearity of both drugs was good, R² value were 0.9996 within their respective concentration ranges. The method was found to be reliable and reproducible with the recovery value of 99.72-102.17% for Linagliptin, whereas %RSD was found to be below 2% for all the precision studies which indicates the reliability of the method, and found to be below 2% for Empagliflozin which indicates recovery value of 99.29-101.20%. The assay results showed acceptable release of drug from the marketed formulation, while the stress degradation studies under acidic, alkaline, oxidative, neutral and photolytic conditions showed good separation of the drug degradation products from the analytes, proving the stability indicating nature of the method. The developed RP HPLC method is simple, rapid, selective, accurate, economical, and can be used for routine pharmaceutical quality control, stability testing and analysis of combined Linagliptin and Empagliflozin formulation.

 

INTRODUCTION:

Empagliflozin is an orally active antidiabetic agent, which has emerged as an important therapeutic agent in the management of T2DM, especially in patients at risk of cardiovascular and renal events. .[8-10] The chemical class sodium–glucose co‑transporter 2 (SGLT2) inhibitors acts by promoting glucosuria and reducing the reabsorption of glucose in the renal proximal convoluted tubules, independent of insulin production and activity. Linagliptin is an orally active highly selective dipeptidyl peptidase‑4 (DPP‑4) inhibitor which improves glycaemic control in adults with T2DM by blocking the rapid degradation of incretin hormones resulting in increases in glucose dependent insulin secretion and decreases in glucagon release and thus reducing FPG and PPG .[13][14]

Empagliflozin and linagliptin provide a rational fixed‑dose combination that targets complementary mechanism of action to provide more thorough glycaemic control in T2DM. The anti-diabetic activity of SGLT2 inhibition (empagliflozin) is insulin-independent, leading to small reductions in body weight and decrease in blood pressure; whereas anti-diabetic activity of DPP‑4 inhibition (linagliptin) is independent of glucose, augmenting incretin activity and improving β‑cell function, with no increase of hypoglycaemia risk when used alone or in addition to insulin or sulfonylureas).[8, 13-14]

Clinical trials and meta‑analyses have shown that fixed‑dose combinations of empagliflozin 10 or 25 mg with linagliptin 5 mg produce significantly greater reductions in HbA1c and FPG compared with either agent alone, with a higher proportion of patients achieving HbA1c below 7% and additional benefits in body‑weight reduction, particularly versus linagliptin monotherapy.[18-20] The combination has demonstrated a safety profile comparable to monotherapy, with the added advantages of once‑daily dosing, reduced regimen complexity, and potential improvements in medication adherence and overall clinical outcomes.[18, 20] Given the high prevalence of T2DM and the need for durable glycaemic control without excessive treatment burden, such fixed‑dose combinations play an important role in optimizing therapy, simplifying treatment, and possibly enhancing cardiovascular and renal protection in appropriate populations.[19-20]

MATERIALS:

Linagliptin and Empagliflozin active pharmaceutical ingredients (APIs) were procured from Swapnroop Drugs and Pharmaceuticals, Aurangabad, India. The marketed formulation, Empaford-L 25/5 tablets, was obtained from Leeford. HPLC-grade methanol, acetonitrile, water, and 0.1% acetic acid (adjusted to pH 4.2 with triethylamine) were purchased from Merck Ltd., India. All chemicals, solvents, and reagents used in the study were of HPLC grade and were used as received without further purification.

METHODOLOGY:

Determination of Absorption Spectra (λmax)

The maximum absorption wavelengths (λmax) of Linagliptin and Empagliflozin were determined using a UV–Visible spectrophotometer. Standard stock solutions of both the drugs were prepared in methanol, diluted appropriately, and scanned from there from 200-400 nm in blank (methanol). The λmax of Linagliptin and Empagliflozin were obtained from their spectra and the overlain spectra were used to obtain the common detection wavelength. The wavelength was chosen for detection as an optimum wavelength in the RP-HPLC method for both analytes because of their spectral features.

HPLC Instrumentation and Chromatographic Conditions

Chromatographic analysis was performed using an Agilent 1100 High-Performance Liquid Chromatography (HPLC) system equipped with a G1310A ISOPUMP, a UV detector, and CHEMSTATION software for data acquisition and processing. Separation was achieved on an Agilent RP-C18 column (4.6 × 100 mm, 2.5 µm particle size) using a mobile phase consisting of methanol and 0.1% acetic acid (pH 4.2 adjusted with triethylamine) in the ratio of 30:70 (v/v). The mobile phase was delivered at a flow rate of 0.7 mL/min, and the analytes were detected at a wavelength of 235 nm. The column temperature was maintained at 25°C, and the injection volume was 20 µL. The HPLC system employed a G1310A ISOPUMP reciprocating pump with a maximum operating pressure of 400 bar, a flow rate range of 0.001–5.0 mL/min, pressure display accuracy of ±5%, four-solvent capability, and a programmable mixing ratio ranging from 0–100%.

Mobile Phase Optimization

Several combinations of methanol and orthophosphoric acid (OPA) solution with different compositions, pH values, and flow rates were used to optimize the chromatographic conditions. Various mobile phase compositions including 90:10, 75:25, 70:30, 80:20, 50:50, 40:60, 30:70, and 25:75 (Methanol:OPA) were investigated using 0.05% and 0.1% OPA with flow rates of 0.7 and 1.0 mL/min. The pH adjustment using triethylamine (TEA) and various detection wavelengths was also tested. Retention time and peak symmetry, theoretical plates, peak resolution and peak shape were used to evaluate the chromatographic performance. Optimized chromatographic conditions were 30% methanol and 70% 0.1% OPA adjusted to pH 40% (flow rate 0.7 mL/min at UV 235 nm) which gave satisfactory separation of Linagliptin and Empagliflozin.

Table 1. Trials for Mobile Phase Optimization

Trial No.	Mobile Phase Composition (v/v)	Flow Rate (mL/min)
1	Methanol 90% + 0.05% Orthophosphoric Acid 10%	0.7
2	Methanol 75% + 0.05% Orthophosphoric Acid 25%	0.7
3	Methanol 70% + 0.05% Orthophosphoric Acid 30%	0.7
4	Methanol 70% + 0.05% Orthophosphoric Acid 30%	1.0
5	Methanol 80% + 0.1% Orthophosphoric Acid 20%	0.7
6	Methanol 80% + 0.1% Orthophosphoric Acid 20%	0.7
7	Methanol 80% + 0.1% Orthophosphoric Acid 20%	0.7
8	Methanol 80% + 0.1% Orthophosphoric Acid 20% (pH adjusted with TEA)	0.7
9	Methanol 50% + 0.1% Orthophosphoric Acid 50% (pH 4.0)	0.7
10	Methanol 40% + 0.1% Orthophosphoric Acid 60% (pH 4.0)	0.7
11	Methanol 30% + 0.1% Orthophosphoric Acid 70% (pH 4.0)	0.7
12	Methanol 25% + 0.1% Orthophosphoric Acid 75% (pH 4.0)	0.7
13	Methanol 30% + 0.1% Orthophosphoric Acid 70% (pH 4.0)	0.7

 

Preparation of Standard Stock Solution

Accurately weighed 10 mg of Linagliptin and Empagliflozin respectively were transferred and dissolved in 100 mL volumetric flasks with methanol and volume was made up to the mark, using the same solvent to obtain the standard stock solution of 100 µg/mL of each drug. Appropriate dilutions were prepared from these stock solutions for method development and validation studies.

Linearity

Linearity of the developed RP-HPLC method was determined by preparing five different levels of standard solution of Linagliptin (5, 10, 15, 20 and 25 µg/mL) and Empagliflozin (10, 20, 30, 40 and 50 µg/mL) with suitable dilutions of stock solution. Concentrations were injected in duplicate separately, with optimized chromatographic conditions. Calibration curves were created by plotting the mean peak area against concentration and the regression equation was calculated with determination of R² value to check the linearity between concentration and detector response.

Accuracy (Recovery Study)

The standard addition method was used to determine the accuracy of the developed method at 80%, 100% and 120% recoveries. The concentration of pure  Linagliptin and Empagliflozin were spiked into pre analysed tablet sample solutions at concentrations of 80, 100 and 120% of the nominal concentration of the drugs. Course wares of both the level were analysed in duplicate under the optimized condition of chromatography and its accuracy has been checked by calculating % recovery, mean, standard deviation (SD) and %RSD.

Repeatability

The RP-HPLC method was found to be repeatable using six replicate preparations of the standard solution (15 µg/mL Linagliptin and 30 µg/mL of Empagliflozin) with the same chromatographic conditions. Each injected peak area values were obtained and further the mean peak area, standard deviation, %RSD were calculated. When %RSD value is below 2% the repeatability of the method is acceptable.

Intraday Precision

To validate the intraday precision, three different concentrations of Linagliptin (5, 15 and 25 µg/mL) and Empagliflozin (10, 30 and 50 µg/mL) were analysed on the same day under the same chromatographic conditions. The peak areas for each concentration were acquired thrice and the mean, standard deviation and %RSD calculated. The accuracy of the method was tested by comparing the analytical results within one day period.

Interday Precision

To find the interday precision, freshly prepared standard solutions of Linagliptin (5, 15 and 25 µg/mL) and Empagliflozin (10, 30 and 50 µg/mL) were analysed on three consecutive days by the optimized chromatographic conditions. Replication was performed three times for each concentration and the arithmetic mean of three replicates, standard deviation and percent relative standard deviation were obtained. In order to evaluate the reproducibility of the method from day to day, the test was performed on several different days in accordance with the ICH.

Limit of Detection (LOD)

The limit of detection (LOD) of the developed RP-HPLC method for Linagliptin and Empagliflozin was determined in accordance with ICH Q2(R2) guidelines using the standard deviation of the response (σ) and the slope (S) of the calibration curve. The LOD was calculated using the equation

The calculated LOD values indicated the lowest concentration of each analyte that could be reliably detected but not necessarily quantified under the optimized chromatographic conditions.

Limit of Quantification (LOQ)

Standard deviation of the response (σ) and the slope (S) of the calibration curve were used to determine the limit of quantification (LOQ) according to the ICH Q2(R2) guidelines. The LOQ were calculated by the equation

where σ is the standard deviation and S is the signal. The acquired LOQ values were the lowest concentrations of Linagliptin and Empagliflozin which could be quantified with acceptable accuracy and precision with the developed RP-HPLC method.

Ruggedness

The ruggedness of the developed RP-HPLC method was determined by comparing two analyst chromatograms of standard solutions (25µg/mL of Linagliptin, 50µg/mL of Empagliflozin) during similar analysis where chromatographic conditions were used. Peak areas obtained were compared and the mean, std and %RSD were calculated to determine the reproducibility of the method under normal laboratory operating conditions.

Robustness

The developed RP-HPLC method was validated for robustness by deliberately making small changes in the chromatographic conditions. To investigate the effect of varying the flow rate from 0.6 mL/min to 0.8 mL/min, the composition of the mobile phase was changed from 69:31 (Water:Methanol) to 71:29 (Water:Methanol) and the detection wavelength was changed from 234 nm to 236 nm, 10 µg/mL of Linagliptin, 20 µg/mL of Empagliflozin were used. Response obtained under each modified condition was evaluated by calculating Mean peak area of the chromatogram peak, Standard deviation and %RSD to check the robustness of the developed analytical method.

Assay of Marketed Formulation

Empaford-L 25/5 containing Linagliptin and Empagliflozin were exactly weighed and the average weight was calculated. Ten milligrams of each of Linagliptin and Empagliflozin was precisely weighed and placed in 100 mL volumetric flasks where the powders are finely ground by sonication and then the flasks are filled to the mark with methanol. Optimized mobile phase was employed for getting appropriate dilutions of 25µg/mL of Linagliptin and 50µg/mL of Empagliflozin after passing through a 0.45µm membrane filter. The optimized HPLC conditions were applied for the injection of the prepared sample solution and the calculation of the assay was carried out by comparing the sample peak areas with the standard peak areas of the same HPLC conditions.

Forced Degradation Studies

Acid Degradation:

The standard drug solution was subjected to acidic degradation by mixing with 0.1N hydrochloric acid (HCl) and reaction was carried out for 1 hour and 2 hours at room temperature to produce acidic degradation product. The solution was then neutralized with a volume of 0.1 N sodium hydroxide (NaOH) solution that was equal to the volume of the injected solution, diluted according to the required strength of the mobile phase, passed through a membrane filter (0.45µm) and introduced into the ion-exclusion HPLC system for analysis after the specified time interval.

Alkaline Degradation:

The standard drug solution was treated with 0.1 N sodium hydroxide (NaOH) for 1 hour and 2 hours to further degrade the drug by acid hydrolysis. After degradation, solution was neutralized with an equal volume of 0.1 N hydrochloric acid (HCL) diluted in the mobile phase and filtered through the 0.45 µm membrane filter and analysed by the optimized RP-HPLC.

Oxidative Degradation:

Three percent, H2O2, was used as the oxidising agent and the studies were carried out by keeping standard drug solution for 1 and 2 hours. The solution was suitably diluted with the mobile phase after degradation period, filtered through a 0.45 µm membrane filter and injected into the chromatographic system, to assess the oxidative degradation.

Neutral Degradation:

The standard drug solution was placed in distilled water for 2 hrs at room temperature (neutral hydrolysis). The degraded solution then was diluted with the mobile phase, passed through a membrane filter of 0.45 µm and was subjected to chromatography on the optimized conditions.

Photolytic Degradation:

The UV degradation studies were carried out by placing the Control drug solution under the action of the UV radiation source for 24 hours. After exposure the solution was diluted with mobile phase, filtered through a membrane filter of 0.45 µm and injected into the RPHPLC system. The resulting chromatograms were then analysed to understand the degree of degradation by the Photolysis and also to validate the method developed as a stability indicating method.

RESULTS AND DISCUSSION

Determination of Absorption Spectra (λ max):

The UV-vis spectra of absorption of Linagliptin and Empagliflozin were measured in methanol in the range of 200–400 nm on UV-vis spectrophotometer. The λmax of the Linagliptin was 235 nm, while that of Empagliflozin was 223 nm. The overlapped spectra of both drugs showed an isosbestic point at 235 nm, this value was selected as a possible detection wavelength for simultaneous RP-HPLC analysis providing adequate sensitivity for both drugs.

Fig 1. Absorption spectra of Linagliptin

Fig 2. Absorption spectra of Empagliflozin

Fig 3. Absorption spectra of Iso-electric point

Mobile Phase Optimization

Different chromatographic conditions involving the proportion of methanol to 0.1% orthophosphoric acid (OPA), conditions of pH and flow rate were tested to obtain good resolution, peak shape and retention time for both the analytes, using RP-HPLC method. When higher proportions of methanol were used (90:10, 75:25 and 70:30, methanol:0.05% OPA), several peaks appeared which could not be resolved and poor selectivity and separation were obtained. Further modifications included changes in the mobile phase composition, adjustment of the pH by triethylamine and the optimization of the wavelength greatly improved the chromatographical performance. The optimized chromatographic conditions were: the mobile phase consisted of 30% MeOH and 70% 0.1% OPA (pH 4.0) at a flow rate of 0.7 mL/min that provided well-resolved peaks with good symmetry for both Linagliptin (T=3.18 min) and Empagliflozin (T=5.84 min). The theoretical plates and peak symmetry obtained for both analytes were found to be acceptable, thus validating the optimized chromatographic conditions for further validation.

Table 2. Chromatographic performance data of mobile phase trials

 

Fig 4. Chromatogram of Trial 13 mobile phase composition

Linearity

Linearity of the developed RP-HPLC method was checked by analysing the concentration range of Linagliptin (5 – 25 µg/mL) and Empagliflozin (10 – 50 µg/mL). Excellent linear relationship was found with Linagliptin with the regression equation: y = 133.70 x + 88.36 and correlation coefficient (R² = 0.9996). The same pattern was observed for Empagliflozin with good linear regression, y = 167.01x + 373.76, R² = 0.9996. The percent relative standard deviation (%RSD) and the standard deviation, obtained at all concentration levels has been satisfactory indicating the good level of proportionality between concentration and peak area and thus suitability of the developed method for the quantitative estimation of both the drugs.

Table 3.  Linearity Data for Linagliptin

 

Table 4.  Linearity Data for Empagliflozin

Concentration (µg/mL)	Area I	Area II	Mean Area	SD	%RSD
10	2030.62	2033.90	2032.26	2.32	0.11
20	3680.06	3678.48	3679.27	1.12	0.03
30	5473.14	5466.03	5469.59	5.03	0.09
40	7020.19	7058.49	7039.34	27.08	0.38
50	8672.69	8729.08	8700.89	39.87	0.46

 

Fig 5. Calibration Curve of Linagliptin

Fig 6.  Calibration Curve of Empagliflozin

Accuracy (Recovery Study)

The method developed was validated by the recovery tests using the standard addition method at 80%, 100% and 120% concentrations and found accurate at 80% concentration to which the result was acceptable. The percentages of recoveries of Linagliptin ranged from ~ 99.72 to 102.17% and Empagliflozin from ~ 99.29 to 101.20%. The %RSD obtained were below the acceptable limit of 2% and indicated a good level of recovery and little interference from formulation excipients. The developed method showed to be accurate and reliable as these findings proved the same.

Table 5.  Summary of Accuracy Studies of Linagliptin

 

Table 6.  Summary of Accuracy Studies of Empagliflozin

Sample (% level)	Conc. of marketed formulation (µg/mL)	Conc. of API added (µg/mL)	Absorbance (Area)	Amount Recovered (µg/mL)	% Recovery	Mean	SD	%RSD
80%	10	8	3372.98	7.96	99.50	99.32	0.25	0.25
			3368.30	7.93	99.15
100%		10	3699.15	9.91	99.13	99.29	0.22	0.22
			3704.42	9.94	99.44
120%		12	4071.15	12.14	101.17	101.20	0.04	0.04
			4072.36	12.15	101.23

Repeatability

The developed RP-HPLC method was tested for repeatability by conducting 6 repeated injections of a standard solution using the same chromatographic conditions. The results obtained by Linagliptin showed a %RSD value of 0.01% and that for the Empagliflozin was 0.18%. The obtained low %RSD were excellent, which suggests very good reproducibility of the analytical procedure and proves the good stability of the method of chromatography developed.

Table 7.  Repeatability for Linagliptin

Concentration (µg/mL)	Mean Area	Amount Found (µg/mL)	% Amount Found	SD	%RSD
15	2123.15	15.22	101.46	0.22	0.01

 

Table 8.  Repeatability for Empagliflozin

Concentration (µg/mL)	Mean Area	Amount Found (µg/mL)	% Amount Found	SD	%RSD
30	5467.83	30.50	101.68	10.08	0.18

 

Intraday Precision

Three different concentrations of Linagliptin (5 µg/mL, 15 µg/mL, 25 µg/mL) and Empagliflozin (10µg/mL, 30 µg/mL, 50 µg/mL) have been analysed and used as a measure of intraday precision. The %RSD for Linagliptin were in the range of 0.12% to 0.46%, while for Empagliflozin the range were 0.20% to 0.28%. All the %RSD obtained during the one day of the analysis was under 2% which reflected that the method had very good precision and reproducibility in the work.

 

 

Table 9.  Intraday Precision for Linagliptin

Concentration (µg/mL)	Mean Area	Amount Found (µg/mL)	% Amount Found	SD	%RSD
5	763.16	5.05	100.94	3.54	0.46
15	2125.54	15.24	101.58	2.83	0.13
25	3463.06	25.24	100.96	4.19	0.12

 

Table 10.  Intraday Precision for Empagliflozin

Concentration (µg/mL)	Mean Area	Amount Found (µg/mL)	% Amount Found	SD	%RSD
10	2054.04	10.06	100.62	5.38	0.26
30	5467.48	30.50	101.67	10.68	0.20
50	8722.32	49.99	99.98	24.04	0.28

 

Interday Precision

The precision of analysis between days was determined by analysing three different concentration levels of freshly prepared standard solutions three days in a row. The %RSD for Linagliptin varied from 0.01% to 0.27% and for Empagliflozin, it was 0.01% to 0.26%.

Table 11.  Interday Precision for Linagliptin

Concentration (µg/mL)	Mean Area	Amount Found (µg/mL)	% Amount Found	SD	%RSD
5	762.21	5.04	100.80	0.11	0.01
15	2125.32	15.24	101.57	5.66	0.27
25	3430.95	25.00	100.00	3.14	0.09

 

Table 12.  Interday Precision for Empagliflozin

Concentration (µg/mL)	Mean Area	Amount Found (µg/mL)	% Amount Found	SD	%RSD
10	2065.30	10.13	101.29	5.33	0.26
30	5479.87	30.58	101.92	0.52	0.01
50	8681.56	49.75	99.50	9.19	0.11

 

Sensitivity (LOD and LOQ)

To evaluate the sensitivity for the developed RP-HPLC method, the limit of detection (LOD) and limit of quantification (LOQ) were determined as per ICH guidelines and justified with the standard deviation of the responses and the slope of calibration curve. Both LOD and LOQ values obtained, for Linagliptin or Empagliflozin were satisfactory enough to enable method to detect the presence of both analytes as well as quantify them accurately in routine quality control/Stability-indicating analysis of very low level of both.

 

LOD and LOQ for Linagliptin

LOD and LOQ for Empagliflozin

Ruggedness

The method was further evaluated for ruggedness by analysing prototype drugs of interest, Linagliptin and Empagliflozin under the similar experimental conditions by different analysts. The percentage relative standard deviations (RSD) for Linagliptin and Empagliflozin were 0.077% and 0.129%, respectively. The small % RSD values showed that the method is rugged and hence the developed analytical method performs consistently and reproducible assays regardless of minor variations in the work.

Table 13.  Ruggedness Study Results of Linagliptin and Empagliflozin

Drug	Conc (µg/mL)	Area I	Area II	Mean Area	Amount Found (Mean, µg/mL)	% Label Claim (Mean)	SD	%RSD
Lina	25	3431.30	3427.64	3429.47	24.99	99.96	0.019	0.077
Emp	50	8640.10	8624.98	8632.54	49.45	98.91	0.064	0.129

 

Robustness

The strength of that RP-HPLC method was tested by deliberately varying the chromatographic conditions such as the flow rate (0.1 mL/min), mobile phase composition (1%) and detection wave length (1 nm). In all the modified conditions, both Linagliptin and Empagliflozin showed good chromatographic performance (see %RSD values below 2%), suggesting that slight variations in the analysis parameters did not noticeably affect changes in the retention time, peak area and peak symmetry. The results obtained were found to be satisfactory and suitable for the routine pharmaceutical analysis and demonstrated the suitability and robustness of the developed method.

 

 

 

Table 14.  Summary for robustness study

Parameter	Drug	Condition	Conc (µg/mL)	Area	Mean	SD	%RSD
Flow Rate Change	Linagliptin	0.6 mL/min	10	1727.26	1712.83	20.41	1.19
				1698.39
		0.8 mL/min		1205.42	1210.32	6.92	0.57
				1215.21
	Empagliflozin	0.6 mL/min	20	4498.13	4479.04	27.00	0.60
				4459.95
		0.8 mL/min		3237.91	3230.67	10.25	0.32
				3223.42
Mobile Phase Change	Linagliptin	69:31 (Water:MeOH)	10	1461.08	1472.80	16.64	1.13
				1484.61
		71:29 (Water:MeOH)		1438.50	1436.06	3.45	0.24
				1433.62
	Empagliflozin	69:31 (MeOH)	20	3806.79	3814.80	11.35	0.30
				3822.84
		71:29 (MeOH)		3828.47	3847.39	26.76	0.70
				3866.31
Wavelength Change	Linagliptin	234 nm	10	1503.43	1509.00	7.84	0.52
				1514.52
		236 nm		1423.01	1424.44	2.02	0.14
				1425.87
	Empagliflozin	234 nm	20	4234.03	4235.70	2.41	0.06
				4237.44
		236 nm		3444.72	3464.52	27.99	0.81
				3484.31

 

Assay of Tablet Formulation

The developed RP-HPLC method was found to be suitable for the assay of the marketed Empaford-L 25/5 tablet. Linagliptin has a mean assay value of 101.10% of the claimed value and Empagliflozin has a mean assay value of 101.22% of the claimed value. The obtained values of standard deviation and %RSD were small and were satisfactory indicating good agreement among replicate injections, ascertaining the developed method's suitability for routine quality control application of combined tablet dosage forms.

Table 15.  Assay Results for Linagliptin

Injection	Area	Amount Found	% Label Claim
I	3477.43	25.35	101.39
II	3457.51	25.20	100.80
Mean ± SD	3467.47 ± 14.09	25.27 ± 0.11	101.10 ± 0.42

 

Table 16.  Assay Results for Empagliflozin

Injection	Area	Amount Found	% Label Claim
I	8833.85	50.66	101.32
II	8816.94	50.56	101.12
Mean ± SD	8825.40 ± 11.96	50.61 ± 0.07	101.22 ± 0.14

 

Fig 7.  Chromatogram of Tablet assay (25 + 50 µg/mL) – Injection 02

Forced Degradation Studies

Acid Degradation

Linagliptin and Empagliflozin were subjected to the acid stress condition (with 0.1 N hydrochloric acid (HCL)) to evaluate the developed RP-HPLC method. Linagliptin also appeared to be moderately susceptible toward acid hydrolysis as 10.38% of the drug was degraded after 1 hour and Empagliflozin was degraded by 5.27% after 1 hour. After 2 h, both drugs were completely degraded (100%), indicating that both were very sensitive to long term acid treatment. All of the degradation peaks were well resolved from the analyte peak without any peak overlaps under the acidic stress environment indicating the specificity and stability indicative property of the developed RP-HPLC method.

Alkaline Degradation

For assessing the alkaline stability of both analytes, alkaline degradation was done using 0.1 N sodium hydroxide (NaOH). Linagliptin also degraded slightly under the alkaline hydrolysis conditions, with 9.87% degradation after 1 hour, whereas Empagliflozin degraded by 3.45% after 1 hour. Linagliptin, on the other hand, was fully degraded (100%) after 2 hrs, and is highly susceptible to long-term alkaline stress. Also, under prolonged alkaline conditions, Empagliflozin was completely degraded. Clear separation of degradation products from the main drug peaks in the chromatograms indicated the developed analytical method is a stability-indicating analytical method.

Oxidative Degradation

Degradation by oxidation has been done with 3% hydrogen peroxide, H₂O₂. Both Linagliptin and Empagliflozin showed good oxidative stability during short-term exposure, showing 3.38% degradation and 4.25%, respectively. Linagliptin degrades by 32.47% after 2 hours as compared to the Empagliflozin degradant that feft only 7.20%, it means Linagliptin is comparatively more susceptible to oxidative degradation than the other compound Empagliflozin. A good resolution of degradants from various peaks was obtained even after the presence of degradation products, showing that the developed RP-HPLC method is specific.

Neutral Degradation

Neutral hydrolysis studies were made of both drugs by keeping them in neutral conditions for two hours. Compared to both drugs, Linagliptin is more stable in neutral conditions with only 0.30% degradation, suggesting excellent stability in neutral condition while Empagliflozin showed 1.07% degradation under neutral condition. The developed method was able to estimate both analytes accurately even after a period of neutral stressful condition where no significant interference from degradation products was observed, proving the method would enhance good resolution of the drug peaks.

Table 17.  Degradation Calculation of Linagliptin (After 1 Hour)

 

Table 18.  Degradation Calculation of Empagliflozin (After 1 Hour)

 

 

 

 

 

Table 19.  Degradation Calculation of Linagliptin (After 2 Hours)

 

Table 20.  Degradation Calculation of Empagliflozin (After 2 Hours)

 

Photolytic Degradation (UV Exposure, 24 Hours)

UV degradation studies were performed by irradiating Linagliptin and Empagliflozin in UV light for 24 hours. Photolytic degradation was tested by exposure of both products, Linagliptin and Empagliflozin to the UV for a period of 24 hours. Linagliptin exhibited 1.41 % degradation whereas Empagliflozin showed only 0.50 % degradation, suggesting that both drugs are highly photolytically stable. The chromatograms showed that the developed RP-HPLC method permitted the complete separation of both degradation products of the drugs from the analytes eliminating the problem of peak purity, and hence appropriate for selective estimation of both drugs along with their degradation products and as a stability indicating analytical method.

Table 20=1.  Photodegradation Calculation of Linagliptin and Empagliflozin (After 24 Hours)

Sr. No.	Drug	Area of Standard	Area of Degraded Sample	% Remaining	Actual % Degradation
1	Linagliptin	2107.287	2077.66	98.59	1.41
2	Empagliflozin	5469.59	5442.13	99.50	0.50

 

Fig 8.  Chromatogram of Photolytic Degradation (UV Light Exposure, 24 h)

CONCLUSION:

Simple, rapid and accurate analysis of Linagliptin and Empagliflozin was successfully developed and validated according to ICH Q2(R2) guidelines from bulk drug and pharmaceutical tablet formulations using RP-HPLC method. Chromatographic separation was carried out with a methanolic mobile phase (30:70 v/v) containing 0.1% orthophosphoric acid at a flow-rate of 0.7 mL/min using UV detector at 235 nm which gives well separated peaks with retention time of 3.18 min for Linagliptin and 5.84 min for Empagliflozin, respectively. The obtained method showed very good linearity over the detection range, the linearity coefficients (R²) for both drugs were 0.9996. The satisfactory accuracy, precision, repeatability, ruggedness, robustness and sensitivity were observed in the validation studies with all the %RSD values staying within the acceptable limit as per ICH guidelines. Results obtained by the assay of marketed formulation were within acceptable limit of 98–102% of labeled claim indicating the suitability of the method for routine quantitative analysis. The forced degradation studies carried out under acidic, alkaline, oxidative, neutral and photolytic stress conditions proved that the method had the ability to cleanly isolate degradation products of the two drugs from the respective drugs and that the developed method is stability indicating. It also showed that both drugs were very prone degradation in the presence of long degradation periods of acid and alkaline conditions, moderately susceptible to degradation in oxidative stress and show good stability under neutral and photolytic conditions. Overall, the developed RP-HPLC method is reliable, selective, reproducible, and can be used for routine quality control analysis, stability testing, formulation development, and regulatory applications with the combined Linagliptin/Empagliflozin pharmaceutical dosage form.

 

 

 

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