A Validated RP-HPLC Method for Simultaneous Estimation of Empagliflozin and Linagliptin in Pharmaceutical Tablet Dosage Form

Type 2 Diabetes Mellitus (T2DM) is one of the most prevalent chronic metabolic disorders globally, with the International Diabetes Federation (IDF) estimating approximately 537 million adults living with diabetes in 2021 — a figure projected to reach 783 million by 2045. The progressive nature of T2DM necessitates multi-drug combination therapy to achieve and sustain optimal glycemic control, driving the development of fixed-dose combinations (FDCs) with complementary mechanisms of action.

Empagliflozin (EMPA), a selective Sodium-Glucose Co-Transporter 2 (SGLT-2) inhibitor, and Linagliptin (LINA), a potent Dipeptidyl Peptidase-4 (DPP-4) inhibitor, represent two mechanistically distinct and complementary antidiabetic drug classes. Their combination in a single tablet (Glyxambi®, Boehringer Ingelheim/Eli Lilly; EMPA 10 mg + LINA 5 mg and EMPA 25 mg + LINA 5 mg) received US FDA approval in February 2015 as the first SGLT-2/DPP-4 inhibitor FDC. SGLT-2 inhibition lowers blood glucose by promoting urinary glucose excretion in an insulin-independent manner, while DPP-4 inhibition attenuates incretin degradation, enhancing postprandial insulin secretion and suppressing the compensatory glucagon elevation induced by SGLT-2 inhibitors.

Accurate and reliable analytical methods for the simultaneous determination of both APIs in the finished dosage form are indispensable for batch release testing, in-process quality control, stability studies, and regulatory compliance. Despite the clinical significance of the Empagliflozin-Linagliptin FDC, the published literature on simultaneous RP-HPLC methods for this combination remains limited. The few available methods exhibit limitations including long run times (>12 min), incomplete robustness evaluation, or lack of comprehensive forced degradation studies.

The present work describes the development and comprehensive ICH Q2(R1)-compliant validation of a simple, rapid, and precise RP-HPLC method for the simultaneous estimation of Empagliflozin and Linagliptin in the combined tablet dosage form, applicable for routine pharmaceutical quality control.

2. DRUG PROFILES

2.1 Empagliflozin (EMPA)

IUPAC Name: D-Glucitol, 1,5-anhydro-1-C-[4-chloro-3-[[4-[[(3S)-tetrahydro-3-furanyl]oxy]phenyl]methyl]phenyl]

Molecular Formula: C??H??ClO?

Molecular Weight: 450.91 g/mol

Melting Point: 145°C–160°C

Solubility: Very slightly soluble in water; slightly soluble in acetonitrile and ethanol; sparingly soluble in methanol.

Half-Life: ~13.1 hours (once-daily dosing)

Approved Dose: 10 mg and 25 mg once daily

Empagliflozin selectively and potently inhibits SGLT-2 in the proximal renal tubules, which mediates approximately 90% of renal glucose reabsorption. Inhibition of SGLT-2 increases urinary glucose excretion in an insulin-independent manner, reducing plasma glucose levels. Beyond glycemic control, Empagliflozin has demonstrated significant cardiovascular and renoprotective benefits. It is commercially available as Jardiance® (10 mg and 25 mg tablets).

2.2 Linagliptin (LINA)

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

Molecular Formula: C??H??N?O?

Molecular Weight: 472.54 g/mol

Melting Point: 190–196°C

Solubility: Soluble in methanol; sparingly soluble in ethanol; very slightly soluble in isopropanol.

Half-Life: ~131 hours (effective DPP-4 inhibition half-life ~12 hours)

Approved Dose: 5 mg once daily (no renal dose adjustment required)

Linagliptin is a xanthine-based DPP-4 inhibitor with picomolar potency (IC?? ~1 nM). DPP-4 inhibition prevents rapid deactivation of incretin hormones GIP and GLP-1, enhancing glucose-dependent insulin secretion and suppressing glucagon. Linagliptin is unique among DPP-4 inhibitors in that it undergoes primarily biliary/fecal elimination (~80%) without significant renal excretion, making it safe across all stages of renal impairment without dose modification. It is marketed as Tradjenta® (5 mg tablets).

3. MATERIALS AND METHODS

3.1  Wavelength Selection

UV absorption spectra of Empagliflozin and Linagliptin (10 μg/mL each in ACN:Water, 50:50 v/v) were recorded between 200 and 400 nm using the Shimadzu UV-1800 spectrophotometer. Empagliflozin exhibited maximum absorbance (λmax) at 227 nm, attributable to the π→π* transition of its aromatic 4-chlorophenyl-phenyl glucoside chromophore system, with a secondary shoulder at approximately 280 nm. Linagliptin displayed strong maximum absorbance at 254 nm due to its highly conjugated xanthine-purine ring system combined with the quinazoline substituent. A detection wavelength of 254 nm was selected for simultaneous detection: it provides maximum sensitivity for Linagliptin (λmax) while affording adequate, quantifiable absorbance for Empagliflozin (~35% of its λmax response). The wavelength is also a standard UV-HPLC wavelength with minimal mobile phase background noise.

3.2 Mobile Phase Optimization

Systematic mobile phase optimization was performed through five sequential trials varying the organic modifier type, buffer composition, and pH:

Table 2: Mobile Phase Optimization Trials

The optimized mobile phase Acetonitrile:0.1% Orthophosphoric acid (60:40 v/v, pH 3.0) provided the best peak symmetry and resolution within a 10-minute run time. The low pH (3.0) effectively suppresses silanol activity on the C18 surface and maintains both analytes in their neutral/protonated forms, ensuring reproducible retention. Acetonitrile was preferred over methanol for its lower UV background below 250 nm and lower viscosity, reducing column backpressure.

3.3 Flow Rate and Column Temperature Optimization

Flow rates of 0.8, 1.0, and 1.2 mL/min were evaluated. At 0.8 mL/min, run time exceeded 13 minutes with decreased peak areas. At 1.2 mL/min, backpressure approached column limits and peaks broadened slightly. A flow rate of 1.0 mL/min yielded optimal peak shape, resolution, and a 10-minute run time within acceptable backpressure (<200 bar). Column temperatures of 25°C, 30°C, and 35°C were also evaluated; 30°C was optimal, providing best peak shapes without excessive temperature-induced changes in selectivity.

3.4 Optimized Chromatographic Conditions

Table 3: Optimized Chromatographic Conditions

4. METHOD VALIDATION

The developed method was validated in accordance with ICH Q2(R1) guidelines for the following parameters: system suitability, linearity, precision (intraday and interday), accuracy, LOD, LOQ, and robustness.

4.1 System Suitability

System suitability was evaluated by injecting the working standard solution (EMPA 100 μg/mL; LINA 50 μg/mL) six consecutive times under the optimized conditions. Peak areas, %RSD, USP plate count, and tailing factor were recorded.

Table 4: System Suitability Parameters

4.2 Linearity

Linearity was established over six concentration levels: 25–150 μg/mL for Empagliflozin and 12.5–75 μg/mL for Linagliptin (n=3 at each level). Peak areas were plotted against concentrations and linear regression analysis was performed.

Table 5: Linearity Summary

Both drugs demonstrated excellent linearity with correlation coefficients ≥ 0.9996, well above the ICH acceptance criterion of R² ≥ 0.999, confirming proportional detector response across the entire analytical range relevant to pharmaceutical dosage form analysis.

4.3 Precision

7.3.1 Intraday Precision (Repeatability)

Table 6: Intraday Precision Results (n=6)

4.3.2 Interday Precision (Intermediate Precision)

Table 7: Interday Precision Results (n=6)

%RSD values for both intraday (EMPA: 0.26%; LINA: 0.44%) and interday precision (EMPA: 0.22%; LINA: 0.31%) were well within the ICH acceptance limit of ≤ 2%, demonstrating excellent method reproducibility.

4.4 Accuracy (Recovery Studies)

Accuracy was evaluated at three spiking levels (50%, 100%, 150% of the nominal concentration; n=3 per level) for each drug.

Table 8: Accuracy Studies — Recovery Data

Mean recovery values ranged from 99.8% to 100.5% for both drugs, confirming the accuracy of the developed method within the ICH acceptance range of 98–102%.

4.5 LOD and LOQ

Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated using the ICH Q2(R1) formula based on calibration curve residual standard deviation (σ) and slope (S): LOD = 3.3σ/S; LOQ = 10σ/S.

Table 9: LOD and LOQ Summary

The low LOD and LOQ values confirm the high sensitivity of the method, making it suitable for detection of trace levels of both analytes including potential degradation products.

4.6 Robustness

Robustness was evaluated by deliberately varying chromatographic parameters within small ranges: flow rate (±0.1 mL/min), mobile phase organic composition (±2%), detection wavelength (±2 nm), and buffer pH (±0.2 units).

Table 10: Robustness Study Results

Under all varied conditions, %RSD values remained below 2%, resolution between EMPA and LINA was consistently maintained above 2.0, and tailing factors were within acceptable limits. These results confirm the robustness of the developed method to small, deliberate changes in chromatographic parameters.

5. ASSAY OF MARKETED FORMULATION

The validated method was applied for the assay of Empagliflozin and Linagliptin in commercially available Glyxambi® tablets (label claim: EMPA 10 mg / LINA 5 mg per tablet). Twenty tablets were weighed and powdered; a quantity equivalent to one tablet weight was extracted with diluent (ACN:Water, 50:50 v/v) by sonication for 15–20 minutes, filtered through 0.45 μm nylon membrane, and appropriately diluted to obtain working concentrations of 100 μg/mL (EMPA) and 50 μg/mL (LINA).

Table 11: Assay of Marketed Formulation (Glyxambi®)

Assay values of 101.4% (EMPA) and 97.7% (LINA) are within the pharmacopoeial acceptance range of 95–105% of labelled claim. The results confirm that the tablet formulation contains the drugs at their stated strengths and that the method is suitable for accurate quantification of both APIs in the presence of tablet excipients.

RESULTS SUMMARY

Table 12: Consolidated Validation Results Summary