Development and Validation of UV Spectrophotometric Methods for the Simultaneous Estimation of Glimepiride and Linagliptin

Diabetes is a chronic disease that occurs when the pancreas does not produce enough insulin or if the body and use of insulin is inefficient. Insulin is a hormone which helps control blood sugar. Without control, diabetes causes hyperglycaemia or high blood sugar, which causes catastrophic damage to many bodies and systems, including neurons and blood vessels, over time.^[1,2]

Glimepiride (GLM): Glimepiride (GLM) is chemically known as 1-[[4-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido) ethyl] phenyl] sulphonyl]-3-trans-(4-methyl cyclohexyl) urea with Molecular Formula and Molecular Weight, C₂₄H₃₄N₄O₅S and 490.6 gm/mole respectively belonging to the Antidiabetic category, shown in Fig. 1. Glimepiride is insoluble in water and soluble in methanol. Mechanism of this drug is, it inhibits ATP-sensitive potassium channel by binding non-specifically to the B sites of both sulfonylurea receptor-1 (SUR1) and sulfonylurea receptor-2A (SUR2A) subunits as well as the A site of SUR1 subunit of the channel to promote insulin secretion from the beta cell.^[3,4]

Figure 1: Chemical Structure of Glimepiride

Linagliptin (LIN): Linagliptin (LIN) is chemically known as 8-[(3R)-3-aminopiperidin-1-yl]- 7-(but-2-yn-1-yl)-3-methyl-1-[(4-methyl quinazolin-2 yl) methyl] purine-2,6-dione with Molecular Formula and Molecular Weight, C₂₅H₂₈N₈O₂ and 472.5gm/mole respectively belonging to the Antidiabetic category, shown in Fig. 2. Linagliptin is freely soluble in water and very soluble in methanol. Mechanism of this drug is, Linagliptin is an inhibitor of DPP-4, an enzyme that degrades the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP).^[5,6]

Figure 2: Chemical Structure of Linagliptin

MATERIALS AND METHODS

Chemicals and Reagents

Analytically pure samples of Glimepiride and Linagliptin were kindly supplied by Exemed Pharmaceuticals, Methanol (UV Grade – Thomas Baker, Mumbai)

Instrument used

Electronic Weighing Balance (Shimadzu 0.1 mg), Ultrasonicator (Athena Technology), UV VIS Spectrophotometer (Shimadzu UV-1800)

Development of Spectrophotometric method and Selection of Solvent: Glimepiride and Linagliptin solution (1000 µg / ml) was prepared with solvent methanol. These solutions were scanned in the UV visible range (200 nm to 800 nm) to determine absorption intensity and absorption wavelength.

Preparation of Standard Solution:

1. Preparation of GLM and LIN standard stock solution (1000 µg/ml): Accurately weigh 10 mg of drug, transfer it to a 10 mL volumetric flask, dissolve in methanol, and make up the volume to the mark with methanol to obtain a 1000 µg/mL solution.

2. Preparation of GLM and LIN working stock solution (100 µg/ml): Pipette 1 mL of the prepared standard stock solution into a 10 mL volumetric flask and make up the volume to the mark with methanol to obtain a 100 µg/mL solution.

First Order Derivative Spectroscopic Method

All zero order spectrum (D0) were converted to first order derivative spectrum (D1) and the overlain first order derivative spectrums of GLM and LIN at different concentration were recorded. The wavelengths reported were 255 nm for GLM (ZCP of LIN) and 232.5 nm for LIN (ZCP of GLM).

Absorption Correction Method

The standard solution of Glimepiride and Linagliptin were diluted with methanol to get the concentration of 2-6 µg/ml and 5-15 µg/ml of respectively. The drugs were scanned in the UV range 200-400 nm. The wavelengths selected were 228 nm for Glimepiride and 294 nm for Linagliptin.

Preparation of Calibration Curve

Calibration Curve for GLM and LIN: Calibration curves were prepared using five different concentrations of standard solutions of GLM (2–6 µg/mL) and LIN (5–15 µg/mL). Each solution was scanned against methanol as blank and the corresponding spectra were recorded. The dA/dλ absorbance was measured at 255 nm for GLM (ZCP of LIN) and at 232.5 nm for LIN (ZCP of GLM). Calibration curves of dA/dλ absorbance versus concentration were plotted, and the respective regression equations were obtained.

Preparation of Sample solution: A synthetic mixture (tablet) equivalent to 2 mg of GLM and 5 mg of LIN was taken into 100 ml of volumetric flask and added 10 ml of methanol, the solution was warmed for 5-10 mins, ultrasonicated for 20 mins, followed by addition of 50 ml methanol and ultrasonicated for 15 min and was makeup upto the mark with methanol. The solution was filtered through Whatmann filter paper no. 41. Thus, resulting solution gave 20 µg/ml of GLM and 50 µg/ml of LIN respectively. From the above solution, 1 ml was pipette out and transferred to 10 ml volumetric flask and volume was made upto mark with methanol in order to give a solution containing GLM (2 µg/ml) + LIN (5 µg/ml). Absorbance of resulting solution was recorded by converting zero order spectra into first order at 255 nm for GLM (ZCP of LIN) and 232.5 nm for LIN (ZCP of GLM).

 Validation of Proposed Method^[7-9]

1. Linearity (n = 5): Linearity was evaluated by analyzing five concentration levels in the range of 2–6 µg/mL for GLM and 5–15 µg/mL for LIN. Calibration curves of dA/dλ absorbance versus concentration were plotted, and the correlation coefficients and regression equations for GLM and LIN were determined.

2. Precision

1. Repeatability (n=6)

An aliquot of 0.4 mL of GLM working stock solution (100 µg/mL) and 1.0 mL of LIN working stock solution (100 µg/mL) were transferred into a 10 mL volumetric flask. The volume was made up to the mark with methanol to obtain a solution containing 4 µg/mL of GLM and 10 µg/mL of LIN. The solution was analyzed six times (n = 6), and %RSD was calculated.

2. Intraday Precision (n=3) Aliquots of 0.3, 0.4, and 0.5 mL of GLM working stock solution (100 µg/mL) and 0.75, 1.0, and 1.25 mL of LIN working stock solution (100 µg/mL) were transferred into separate 10 mL volumetric flasks. The volume was made up to the mark with methanol to obtain solutions containing 3, 4, and 5 µg/mL of GLM and 7.5, 10, and 12.5 µg/mL of LIN. Each solution was analyzed three times (n = 3) on the same day at short time intervals, and %RSD was calculated.
3. Interday Precision (n=3) Aliquots of 0.3, 0.4, and 0.5 mL of GLM working stock solution (100 µg/mL) and 0.75, 1.0, and 1.25 mL of LIN working stock solution (100 µg/mL) were transferred into a series of 10 mL volumetric flasks. The volume was made up to the mark with methanol to obtain solutions containing 3, 4, and 5 µg/mL of GLM and 7.5, 10, and 12.5 µg/mL of LIN. Each solution was analyzed three times (n = 3) on three different days, and %RSD was calculated.

3. Accuracy (n=3)   

Accuracy of the method was assessed by standard addition method at different concentration levels i.e. 80%, 100% and 120%) and % recoveries were computed.

% Recovery = A – B/ C,

Where, A = Total amount of drug estimated

B = Amount of drug found on pre analysed basis

C = Amount of Pure drug added

4. LOD and LOQ 

Detection limit and quantitation limit were determined based on the standard deviation of y-intercepts of calibration curves and average slope of calibration curves.

LOD = 3.3 × S.D./Slope

LOQ = 10 × S.D./Slope

RESULT AND DISCUSSION:

FIRST ORDER DERIVATIVE METHOD:

Figure 1: Overlain Spectra of GLM (2 µg/ml), LIN (5 µg/ml) and MIXTURE  (2 + 5 µg/ml)

Figure 2: Zero Order Spectra of GLM (2-6 µg/ml)

Figure 3: Zero Order Spectra of LIN (5-15 µg/ml)

Figure 4: ZCP of GLM at 232.5 nm

Figure 5: ZCP of LIN at 255 nm

Figure 6: Overlain Spectra of GLM (2-6 µg/ml) and LIN (5-15 µg/ml)

Validation of proposed UV method:

1. Linearity: The linearity for GLM and LIN was found to be in the range of 2-6 µg/ml and 5-15 µg/ml respectively. Linearity data for GLM at 255 nm and LIN at 232.5 nm.

Table 1: Linearity of GLM at 255 nm (ZCP of LIN)

Figure 7: Calibration Curve of GLM at 255 nm (ZCP of LIN)

Table 2: Linearity of LIN at 232.5 nm (ZCP of GLM)

Figure 8: Calibration Curve of LIN at 232.5 nm (ZCP of GLM)

2. PRECISION

1. Repeatability (n=6):

The data of Repeatability for GLM at 255 nm (ZCP of LIN) and LIN at 232.5 nm (ZCP of GLM) is shown in table.

Table 3: Repeatability data for GLM and LIN

2. Intraday Precision (n=3):

The data of Intraday Precision for GLM at 255 nm (ZCP of LIN) and LIN at 232.5 nm (ZCP of GLM) is shown in table.

Table 4: Intraday Precision for GLM at 255 nm

Table 5: Intraday Precision for LIN at 232.5 nm

c) Interday Precision (n=3):

The data of Interday Precision for GLM at 255 nm (ZCP of LIN) and LIN at 232.5 nm (ZCP of GLM) is shown in table.

Table 6: Interday Precision for GLM at 255 nm

Table 7: Interday Precision for LIN at 232.5 nm

3. Accuracy:

Accuracy of the proposed method was assured by performing recovery study from synthetic mixture at three levels by standard addition method. Percentage recovery for GLM and LIN at 232.5 nm and 255 nm were obtained respectively. The result is depicted in table. Recovery was found to be in the limit of 98-102%. 

Table 8: Determination of accuracy of GLM and LIN

4. Analysis of synthetic mixture:

Table 9: Determination of Assay of GLM and LIN

ABSORPTION CORRECTION METHOD: 

Selection of wavelength for estimation of Glimepiride and Linagliptin: 

Figure 9: Overlain Spectra of GLM (2-6 µg/ml) and LIN (5-15 µg/ml)

Validation of proposed UV method: 

1. Linearity: 

The linearity range for GLM and LIN was found to be in the range of 2-6 µg/ml and 5-15 µg/ml respectively. Linearity data for GLM at 228 nm and LIN at 294 nm.

Table 10: Linearity of GLM at 228 nm

Figure 10: Calibration Curve of GLM at 228 nm

Table 11: Linearity of LIN at 294 nm

Figure 11: Calibration Curve of LIN at 294 nm

2. PRECISION

1. Repeatability (n=6):

The data of Repeatability for GLM at 228 nm and LIN at 294 nm is shown in table.

Table 12: Repeatability data for GLM and LIN

2. Intraday Precision (n=3):

Table 13: Intraday Precision for GLM at 228 nm

Table 14: Intraday Precision for LIN at 294 nm

c) Interday Precision (n=3):.

Table 15: Interday Precision for GLM at 228 nm

Table 16: Interday Precision for LIN at 294 nm

3. Accuracy:

Accuracy of the proposed method was assured by performing recovery study from synthetic mixture at three levels by standard addition method. Percentage recovery for GLM and LIN at 228 nm and 294 nm were obtained respectively. The result is depicted in table. Recovery was found to be in the limit of 98-102%. 

Table 17: Determination of accuracy of GLM and LIN

4. Analysis of synthetic mixture: 

Table 18: Determination of Assay of GLM and LIN