Method Development and Validation for Estimation of Dapaglifloizin and Linagliptin in Combined Dosage form by UV Spectrophotometry

The most widespread form of diabetes is type 2 diabetes mellitus that is affecting millions of people across the globe.^[1] Metformin is conventionally the initial oral antidiabetic agent of choice in the management of Type 2 diabetes mellitus (T2DM) based on its many years proven track record of effectiveness, cost, neutrality with respect to weight gain and other positive safety profile.^[2,3] However, as the condition advances, metformin monotherapy is no longer effective enough. Metformin is normally used together with other medications to treat this condition and to improve blood sugar levels.^[4-6] In this aspect, a fixed-dose combination of two categories of anti-diabetic agents with complementary mechanisms of action lowers the dose and the chances of dose-related adverse effects. It is also possible that this strategy will enable the use of individual antidiabetic agents and thereby minimize the number of pills, improve adherence, and attain target HbA1c sooner compared with monotherapy. ^[7-9]

Dipeptidyl peptidase 4 (DPP-4) and sodium-glucose cotransporter-2 (SGLT2) inhibitors used as a combination therapy may represent an effective approach to patients who do not attain sufficient control with metformin or to patients intolerant to metformin.^[10]. As an inhibitor of SGLT2, dapagliflozin (DGF) in the joint protocol has the potential not only to reduce the level of blood sugar by inhibiting renal glucose reabsorption but also to cause a decrease in the number of hypoglycemic episodes since it does not rely on insulin secretion. Meanwhile, DGF possesses the benefit of both weight reduction and the decreased risk of endpoint events, which have been popular in the overweight T2DM population.^[11] Linagliptin, as a DPP-4 inhibitor, has not only the effect of enhancing insulin levels secretion but also the effect of controlling blood sugar level through inhibition of glucagon levels secretion. Linagliptin, in comparison to other DPP-4 inhibitors, is primarily excreted via nonrenal routes. Therefore, patients with impaired renal functions do not require dosage modifications.^[12,13] The synergistic mechanism of action and the good tolerance profiles of the two drug classes make them appropriate choice of treatment when used in combination therapy with any of the already existing glucose-lowering agents including insulin.^[9]

Although the efficacy of LGP and DGF is proved, a large analytical method gap exists to quantify these drugs simultaneously in combination therapy. A substantial literature search was conducted which indicated the existence of numerous ways of the estimation of DGF separately with the help of analysis methods like UV Spectroscopy, RP-HPLC method^[14,15] or in combination with other drugs like metformin or saxagliptin or others with the help of analysis methods like HPLC, RP-HPLC, HPLC-PDA, RP-UPLC,HPTLC, etc.^[16-23] Equally, the estimation of LGP alone is also described in many ways such as HPTLC, UV Spectroscopy^[24-26] or combined with other drugs like metformin or empagliflozinor other drugs using analytical techniques such as HPLC-UV, HPTLC, RP-HPLC.^[19,27-33] Out of the literature review it was observed that methods such as HPLC, HPLC-PDA, RP-HPLC, HPTLC are only a few methods to estimate DGF and LGP in combined dosage form.^[34-37] There is only a single UV spectrophotometric procedure that is available but it is not applicable to the combined dosage form, it is only applicable to the API.^[38] Thus. the objective of the current work was to establish and to validate two easy, fast and robust UV methods to determine DGF and LGP simultaneously in combined dosage form.

MATERIALS AND METHODS

The reference samples of DGF and LGP were procured as gift sample from Morepen Labs Ltd., Parwanoo, Himachal Pradesh, India. The commercial formulation, DAPAVEL-L, marketed by Intas Pharmaceuticals Ltd., India(tablets containing 10 mg of DGF and 5 mg of LGP) was purchased from the local market. Throughout the experiment, analytical-grade materials and fresh purified distilled water was used.

Preparation of Standard Stock Solution

Accurately weighed quantity of 100 mg of DGF and 100 mg of LGP were transferred into 100 mL volumetric flasks individually containing 20 mL of methanol and the volume was made up to the mark with distilled water. These were stock solutions of strength of 1000 μg mL^-1 of both DGF and LGP.

Preparation of Working Standard Solution

From standard stock solutions of DGF and LGP, 10 mL solution was transferred in to 100 mL volumetric flasks and diluted up to mark with distilled water individually. These were stock solution of strength of 100 μgmL^-1 of both DGF and LGP. Aliquots from these secondary stock solutions were appropriately diluted with distilled water to obtain working standard solutions of concentration 10 µg mL^-1 of both the drugs.

Selection of Wavelength

Simultaneous Equation Method (Method I)

The scanning of working standard solutions of DGF and LGP were carried out using Shimadzu UV1800 Double Beam UV-Visible Spectrophotometer in the range of 200–400 nm against distilled water as a blank. The maximum absorbance (λ_max) of DGF and LGP was detected at 222.5 nm (λ₁) and 227.5 nm (λ₂), respectively (Figure 1 and 2).

Figure 1.UV spectrum of dapagliflozin

Figure 2.UV spectrum of linagliptin

AUC Method (Method II)

The area under the curve (AUC) technique is a spectrophotometric technique that is utilized in the simultaneous analysis of mixtures of components especially where the mixtures have broad spectra or no sharp peaks. The AUC approach is based on the principle that the area of a part of the spectrum of a mixture is proportional to the concentration of a given component in that area.

In this method, wavelength range 212.5 nm (λ₃)to 232.5 nm (λ₄) was selected for estimation of DGF and 217.5 nm (λ₅) to 237.5 nm (λ₆) was selected for estimation of LGP.

Preparation of Calibration Curve

Method I

From the secondary standard solutions of DGF (100 µgmL^-1) and LGP (100 µg mL^-1), aliquots of 5,10, 15, 20, 25 and 30 mL were transferred into series of 100 mL volumetric flask and diluted up to mark with distilled water. These were solutions of 5-30 µg mL^-1of both the drugs. The corresponding absorbance were measured at 222.5 nm (λ₁) and 227.5 nm (λ₂). Calibration curves were plotted by taking concentration on x-axis and absorbance on y-axis which showed straight lines. Absorptivity (a) for both the drugs were calculated by the formula:

a = A/C

Where, A= absorbance, C= concentration of analyte in g100 mL^-1

Method II

From working standard solution of DGF (100 µg mL^-1) and LGP (100 µg mL^-1), aliquots of 5, 10, 15, 20, 25 and 30 mL were transferred separately into series of 100 mL volumetric flasks and diluted up to mark with distilled water. This yielded solutions of 5, 10, 15, 20, 25 and 30 µg mL^-1 of both the drugs. The corresponding AUC of all the solutions of DGF were calculated in the range 212.5 nm (λ₃) to 232.5 nm (λ₄) and AUC of all the solutions of LGP were calculated in the range 217.5 nm (λ₅) to 237.5 nm (λ₆). Calibration curves were plotted by taking concentration on x-axis and AUC on y-axis which showed a straight line.

Preparation of Test Solution for Assay Determination

Dapagliflozin/Linagliptin is used in the ratio of 10/5 mg for treatment of diabetes. Ten tablets of Dapavel-L 10/5 tablets were weighed and powdered in a mortar pestle and powder equivalent to 50 mg of DGF was taken into a 50 mL volumetric flask containing 10 mL methanol. The contents of the flask were sonicated for 15 min. to completely dissolve the active ingredients. The volume was made up to mark with distilled water and the solution filtered usingWhatman filter paper no. 41in order to obtain test stock solutionthat correspond to 1000 μgmL^-1 of DGF and 500 μgmL^-1 of LGP. An aliquot of this solution (2 mL) was pipetted into a 100 mL volumetric flask and the volume was brought up to the mark with distilled water. This test solution containing working concentrations of 20 µgmL^-1 DGF and 10 µgmL^-1 LGP was analyzed for assay determination.

Analysis of Marketed Formulation

Method I

When two absorbing drugs are present in a sample and each absorbs at λ_max of the other, it could be possible to determine both drugs using simultaneous equation technique. Two wavelengths selected for the development of the simultaneous equation were 222.5 nm and 227.5 nm. The absorptivity values determined for DGF were 449.78 (ax₁), 424.86 (ax₂) and for LGP were 445.14 (ay₁) and 497.42 (ay₂), respectively at 222.5 nm and 227.5 nm. These values were the means of six estimations. The absorptivity data are shown in Tables 1 and 2.

Table 1.Absorptivity data for dapagliflozin

Table 2.Absorptivity data for linagliptin

Where, A₁ and A₂ are absorbance of mixture at 222.5 nm and 227.5 nm;

C_x and C_y are the unknown concentration of DGF and LGP, respectively in sample solution;

• ax₁ = Absorptivity of DGF at λ₁
• ay₁ = Absorptivity of LGP at λ₁
• ax₂ = Absorptivity of DGF at λ₂
• ay₂ = Absorptivity of LGP at λ₂

Method II

The area and absorptivity values were calculated at each wavelength range and the concentration of both the drugs was calculated from the equations:

A₁ = ax₁ C(x) + ay₁ C(y)        (λ₃−λ₄) nm      (3)

A₂ = ax₂ C(x) + ay₂ C(y)        (λ₅−λ₆) nm       (4)

C_(x) = [A₁ × ay₂−A₂ × ay₁]/[a_X1 × a_Y2 − a_X2 × a_Y1]                 (5)

C_(y) = [A₂ × ax₁−A₁ × ax₂]/[a_X1 × a_Y2−a_X2 × a_Y1]                   (6)

where,

• ax₁ and ax₂ are absorptivities of DGF at (λ₃-λ₄) and (λ₅-λ₆) respectively.
• ay₁ and ay₂ are absorptivities of LGP at (λ₃-λ₄) and (λ₅-λ₆) respectively.
• A₁ and A₂ are AUC of the formulation at (λ₃-λ₄) and (λ₅-λ₆) respectively.
• C_(x) and C_(y) are the concentration of DGF (x) and LGP (y), respectively.

Method Validation

Linearity and Range

Linearity was studied by preparing standard solutions of DGF and LGP at six different concentrations i.e. 5-30 µg mL^-1.

Method I: For each solution, the absorbance of both the drugs was measured at both the wavelengths 222.5 (i.e. λ_max of DGF) and 227.5 nm (i.e. λ_max of LGP). The absorbance verses concentration calibration curves were plotted. Linear regression analysis showed the linearity of the absorbance responses versus concentrations.

Method II: For each solution of DGF, the AUC was calculated in the range 212.5 nm to 232.5 nm and for LGP, AUC was measured in the range 217.5 to 237.5 nm. The calibration curves of AUC versus concentration were plotted.

Precision

The precision of the proposed method was assessed as repeatability, intra-day precision and inter-day precision.

Repeatability was performed by applying six replicates of sample analysis. 5 mL of secondary stock solution (100 µgmL^-1) of LGP standard was transferred to a 100 mL volumetric flask. 10 mL of secondary stock solution of DGF standard (100 μgmL^-1) was transferred to the above 100 mL volumetric flask. The volume was adjusted up to mark with distilled water to get 10 μgmL^-1 solution of DGF and 5 μgmL^-1 solution of LGP.

For intermediate precision, aliquots of 5, 10 and 15 mL of secondary stock solutions (100 µgmL^-1) of LGP were transferred to a series of 100 mL volumetric flask. Aliquots of 10, 20 and 30 mL of secondary stock solution of DGF (100 μgmL^-1) were respectively transferred to the same series of 100 mL volumetric flask. The volume was adjusted up to mark with distilled water to get 10, 20, and 30 μgmL^-1 solutions of DGF and 5, 10 and 15 μgmL^-1 solutions of LGP. Intra- day and inter-day precision of the proposed method was conducted by analyzing the corresponding responses triplicate on the same day and on three different days, respectively to the above solutions and the results are expressed in relative standard deviation (RSD).

Accuracy

Recovery studies were carried out by standard addition method. The standard addition method was performed at three concentration levels in triplicate at 50%, 100% and 150%. All the dilutions were prepared from 100 µgmL^-1 of standard and sample stock solutions. A known amount of standard DGF (5, 10 and 15 µgmL^-1) and LGP (2.5, 5 and 7.5 µgmL^-1) similar to 50%, 100% and 150% of the label claim were added to test solution of DGF (10 µgmL^-1) and LGP (5 µgmL^-1), respectively. The solutions were analyzed in triplicate at each level as per the proposed method. Each recovery was made three times and average value was considered. The percent recovery at each level was calculated.

LOD and LOQ

The LOD and LOQ were estimated from the set of six calibration curves used to determine method linearity by using the following equations:

LOD = 3.3* σ/S and

LOQ = 10* σ/S

Where,

σ = the standard deviation of y- intercepts of regression lines of six calibration curves,

S = the average of the slopes of six calibration curves.

RESULTS AND DISCUSSION

Method Validation

Linearity

The response for the both the drugs was found to be linear in the concentration range of 5-30 µgmL^-1 for both the methods with good linearity (correlation coefficient, r² more than 0.99) (Figure 3,4 and5).

The linearity results are given in Tables 3 to 6. Correlation coefficients for both the drugs at all the wavelengths studied were found to close to 1 which proved the good linearity.

Table 3.Linearity data of linagliptin and dapagliflozin at l₁

*Average of six determinations

Figure 3. Standard plots of linagliptin for method I

Table 4.Linearity data of linagliptin and dapagliflozin at l₂

*Average of six determinations

Figure 4. Standard plots of dapagliflozin for method I

Table 5.Linearity data of dapagliflozin and linagliptin for Method II

*Average of six determinations

Figure 5. Standard plots of dapagliflozin and linagliptin for method II

Table 6.Linearity results of dapagliflozin and linagliptin for method II

Precision

The %RSD of repeatability was found to be less than 2% for both the developed methods (Table 7). The %RSD of intraday and interday precision was also found to be less than 2% for both the methods, thus, confirming precision of the developed methods (Tables 8 and 9).

Table 7.Repeatability test of dapagliflozin and linagliptin

Table 8.Intraday precision test of dapagliflozin and linagliptin

Table 9.Interdayprecision test of dapagliflozin and linagliptin

Accuracy of the method was confirmed by recovery study from marketed formulation at three level of standard addition. Percentage recovery for DGF was in range of 97.03 – 102.22 %, while for LGP, it was found to be in range of 96.88 – 102.02 % (Tables 10to 13) which showed that the developed methods were accurate. The %RSD was also found to be less than 2% in both the methods and thus, confirming the accuracy of result.

Table 10.Accuracy data of dapagliflozin for method I

Table 11.Accuracy data of linagliptin for method I

Table 12. Accuracy data of dapagliflozin for method II

Table 13. Accuracy data of linagliptin for method II

Table 14 demonstrates the LOD and LOQ values calculated by standard formula provided in ICH guidelines. The low values of LOD and LOQ in both the methods indicated that predicted methods are very sensitive.

Table 14. LOD and LOQ data

The developed methodswere applied to test sample solution preparation. The % assay of DGF and LGP was 100.843 ± 1.869% and 100.277 ± 1.913%, respectively of the labelled amount by method I (Table 15). Whereas, the % assay of DGF and LGP was found to be 99.73 ± 1.611 % and 100.42 ± 1.645 %, respectively of the labelled amount by method II (Table 16).These methods were successfully used by several researchers for simultaneous estimation of various drugs.^[39-45]

Table 15. Analysis of test solution by method I

Table 16. Analysis of test solution by method II

The statistical methods were used to analyze the data of the assays to identify the effect of the two projected approaches. The statistical significance of the differences between the two different approaches was compared using one-way ANOVA. The two tests were established at p<0.05. Table 17 illustrates the results of one-way ANOVA and it was observed that the developed procedures were not substantially different between one another.^[46]

Table 17. Statistical comparison of assay results utilizing one way ANOVA