Diabetes is a chronic condition associated with abnormally high levels of sugar (glucose) in the blood. The production of insulin by the pancreas lowers blood glucose level. Due to the absence or insufficient production of insulin causes diabetes. There are two types of diabetes which are referred to as type 1 and type 2 [1]. Formerly names for these conditions were insulin-dependent and non-insulin-dependent diabetes, or juvenile onset and adult onset diabetes. The symptoms of diabetes include increased urine output, thirst, hunger, and fatigue. Diabetes is diagnosed by blood sugar (glucose) testing. The number of individuals affected by diabetes is continuing to increase worldwide; the need for effective management assumes ever greater urgency [2]. Newer classes of medications, particularly those which work via the incretin pathway, achieve glucose lowering and minimizing risks associated with more traditional therapies. Ideally, combination therapies should be well tolerated, convenient to take, have few contraindications, have a low risk of hypoglycemia and weight gain, and be reasonably effective over both the short and long term such as the combination of metformin (MF) and the dipeptidyl peptidase-4 (DPP- 4) inhibitor alogliptin (ALG). The chemical structure of metformin and alogliptin were represented in Figures 1 and 2 respectively. Alogliptin [3-5] is a selective, orally-bioavailable inhibitor of enzymatic activity of dipeptidyl peptidase-4 (DPP-4). DPP-4 inhibitors represent a new therapeutic approach to the treatment of type 2 diabetes that functions to stimulate glucose dependent insulin release and reduce glucagons levels. This is done through inhibition of the inactivation of in cretins, particularly glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP). Alogliptin inhibits dipeptidyl peptidase 4 (DPP- 4), which normally degrades the incretin glucose-dependent insulin tropic polypeptide (GIP) and glucagon like peptide 1 (GLP-1), thereby improving glycemic control [3-5]. Metformin hydrochloride (MTF) [6-9] (C₄H₁₁N₅.HCl) is 1:1 dimethylbiguanidine monohydrochloride is an anti-diabetic drug from the biguanide class of oral Hypoglycaemic agents, given orally in the treatment of non-insulin-dependent diabetes mellitus. The main action of Metformin HCl is in increasing glucose transport across the cell membrane in skeletal muscle. Several analytical methods based on UV [9,10], Spectroflourimetry [11,12], Reverse Phase-HPLC [13-23], LC-MS/MS [24-27], HPTLC [28] were reported for the determination of metformin either in alone or in combination with other drugs. Although literature survey reveals that no methods were reported for metformin (MTF) and alogliptin (ALG) in combination form.

Figure 1: Chemical structure of Metformin HCl.

Figure 2: Chemical structure of Alogliptin.

Chemicals and reagents

The reference sample of metformin and alogliptin were supplied by M/s Pharma Train, Hyderabad. HPLC grade water (prepared by using 0.45 Millipore Milli-Q) was procured from Standard Reagents, Hyderabad. HPLC grade acetonitrile was purchased from Merck, Mumbai. The chemicals used for preparation of buffer include sodium dihydrogen ortho phosphate (Finar Chemicals, Ahmedabad), and orthophosphoric acid (Standard Reagents, Hyderabad). 0.45 μm membrane filters (Advanced Micro Devices Pvt. Ltd., Chandigarh, India) were used for filtration of various solvents and solutions intended for injection into the column [29-32].

Instrumentation

A Waters Alliance 2695 separation module equipped with a 2487 UV detector was employed throughout this study. Column that was employed in the method was X-Terra C18 (4.6 × 150 mm, 3.5 μm, Make: ACE). The samples were injected with an automatic injector. The 20 μL volume of sample was injected. The input and output operations of the chromatographic system were monitored by Waters Empower software. The flow rate selected was 1.0 mL per min. The detection was done at 235 nm. The temperature and run time was monitored at 25°C and 8.0 min respectively. The ultra violet spectra of the drugs used for the investigation were taken on a Lab India UV 3000 spectrophotometer for finding out their λ_max values. Solubility of the compounds was enhanced by sonication on an ultra sonicator. (Model: Power Sonic 510, Hwashin Technology). All the weighings in the experiments were done with an Afcoset electronic balance. The HermLe microlitre centrifuge Z100 (model no 292 P01) was used for the centrifugation process and Remi equipments (model no- CM101DX) Cyclomixer was used.

Glassware

All the volumetric glassware used in the study was of Grade A quality Borosil.

Preparation of sodium phosphate buffer (pH 4.0)

The buffer solution was prepared by dissolving 2.5 g of sodium dihydrogen ortho phosphate in 900 mL of HPLC grade water in a 1000 mL clean and dry flask. The mixture was stirred well until complete dissolution of the salt. The volume was made upto the mark with water. The pH was adjusted to 4.0 with 1% ortho phosphoric acid.

Preparation of mobile phase

The mobile phase was prepared by mixing 700 mL phosphate buffer (pH 4.0) and 300 mL of acetonitrile in a 1000 mL clean and dry flask. The resultant mobile phase was filtered through a 0.45 μm membrane filter (Advanced Micro Devices Pvt. Ltd., Chandigarh, India) under vacuum. The resultant mobile phase was degassed in an ultra sonicator for 5 min.

Preparation of diluent

The diluent was prepared by mixing phosphate buffer (pH 4.0) and acetonitrile (HPLC grade) in the ratio of 70:30 v/v. This solution was used to dilute the drug solutions in the study.

Preparation of standard solution of metformin and alogliptin

10 mg metformin was weighed accurately and transferred into a 10 mL clean and dry volumetric flask. Initially, the drug was mixed with 7 mL of diluent. The solution was sonicated for 15 min for complete dissolution of the drug. The final volume was made up to the mark with the same solvent. Similarly, about 10 mg alogliptin was weighed accurately and transferred into a 100 mL clean and dry volumetric flask. Initially, the drug was mixed with 70 mL of diluent. The solution was sonicated for 15 min for complete dissolution of the drug. The final volume was made up to the mark with the same solvent. From the above prepared stock solutions 5.0 mL of metformin and 1.25 mL of alogliptin were pipetted out into a 10 mL clean and dry volumetric flask and it was diluted up to the mark with diluent. This mixed stock solution contains 500.0 μg/mL of metformin and 12.5 μg/mL of alogliptin.

Spiking of metformin and alogliptin into plasma and their extraction from plasma (By precipitation method)

From the above prepared mixed stock solution (500.0 μg/mL of metformin and 12.5 μg/mL of alogliptin), 0.5 mL was pipetted out and spiked into 0.5 mL of plasma in a polypropylene tube (Torson’s). Then the tube was cyclo mixed for 5 min. Then 1.0 mL of acetonitrile was added to the tube and centrifuged for 20 min at 3000 rpm. Further the supernatant liquids were collected in another Eppendorf tube and 20 μL supernatant was injected into the analytical column.

Selectivity

An aqueous mixture of metformin and alogliptin (500.0 μg/ mL of metformin and 12.5 μg/mL of alogliptin) was prepared and injected into the column and the retention times were checked and any interference at the retention times was checked by comparing the response in the blank. No interference was observed at the retention times for metformin and alogliptin extracted from plasma [33-38]. The method was found to be precise and specific. A typical chromatogram of metformin and alogliptin in plasma is shown in Figure 3.

Figure 3: A typical chromatogram of Metformin and Alogliptin standard drugs in plasma.

Sensitivity

To determine the sensitivity in terms of LLOQ, ‘Lower Limit of Quantification’ where the response of LLOQ must be at least five times greater than the response of interference in blank matrix at the retention time of the analyte(s). The LLOQ obtained by the proposed method were 5.936 and 1.983 μg/mL for metformin and alogliptin respectively.

Precision

To check the intra and inter-day variations of the method, solutions containing 500.0 μg/mL of metformin and 12.5 μg/mL of alogliptin were subjected to the proposed HPLC method of analysis and results obtained were noted. The precision of the proposed method i.e., the intra and inter-day variations in the peak areas of the drugs solutions in plasma were calculated in terms of percent relative standard deviation (RSD) and the results are represented in Tables 1, 2, 3 and 4. A statistical evaluation revealed that the percentage relative standard deviation of the drugs at linearity level for 6 injections was less than 2.0. Typical chromatogram of metformin and alogliptin in plasma for intra and inter-day precision are shown in Figures 4 and 5.

Table 1: Intra-day precision of the proposed method for Metformin in plasma.

Table 2: Intra-day precision of the proposed method for Alogliptin in plasma.

Table 3: Inter-day precision of the proposed method for Metformin (on three consecutive days n=6) in plasma.

Table 4: Inter-day precision of the proposed method for Alogliptin (on three consecutive days n=6) in plasma. *Average of Six injections.

Figure 4: Typical chromatogram of metformin and alogliptin in plasma for intraday precision.

Figure 5: Typical chromatogram of metformin and alogliptin in plasma for interday precision.

Accuracy

To determine the accuracy of the proposed method, recovery studies were carried out by analyzing (8.0, 10.0, 12.0 mg of metformin and alogliptin) of pure drugs. The solutions were suitably diluted at linearity level (500.0 μg/mL of metformin and 12.5 μg/mL of alogliptin). Then each dilution was injected thrice (n=3). The percent recoveries of the drugs were determined. The results are shown in Tables 5 and 6.

Table 5: Accuracy data of the proposed method for Metformin in plasma.

Table 6: Accuracy data of the proposed method for Alogliptin in plasma.

Linearity

In order to find out the linearity range of the proposed HPLC method in plasma, curves were constructed by plotting peak areas obtained for the analyte against their concentrations. A good linear relationship (r²=0.997) was observed between the concentrations of metformin and alogliptin and their corresponding peak areas. The relevant regression equations were y=1192.x-38173 for metformin (r²=0.997) and y=2802x-1113 for alogliptin (r²=0.998) (where y is the peak area and x is the concentration of metformin and alogliptin (μg/ mL)). The slope, intercept and the correlation coefficient of the plots are shown in Tables 7 and 8. The linearity ranges for metformin and alogliptin and their corresponding graphs are shown in Figures 6 and 7.

Table 7: Linearity range of Metformin in plasma.

Table 8: Linearity range of Alogliptin in plasma.

Figure 6: Calibration curve for Metformin in plasma.

Figure 7: Calibration curve for Alogliptin in plasma.

Stability

All stability determinations used a set of samples prepared from a freshly made stock solution of the analyte in the appropriate analytefree, interference-free biological matrix [39]. The stock solutions of the analyte for stability evaluation were prepared in an appropriate solvent at known concentrations. To test the stability of the drug extract, it was subjected to

a) Freeze and thaw stability at -20°C ± 2°C,

b) Short term stability for period of 24 hours stored at room temperature,

c) Long term stability for period of 15 days stored at 4°C.

Similar to the preparation of the standard preparation, the above samples were spiked into the plasma and extracted and collected in vial and injected into HPLC system. All the stability samples compared against the standard stock solution assessed for stability. The results are presented in Tables 9 and 10 (the figures in the table are in peak area units). Typical chromatograms for standard samples, freeze and thaw stability samples, short term stability samples and long term stability samples were represented in Figures 8, 9, 10 and 11.

Table 9: The Stability data for Metformin in plasma.

Table 10: The Stability result for Alogliptin in plasma.

Figure 8: Typical chromatogram for standard samples in plasma for stability studies.

Figure 9: Typical chromatogram for freeze thaw samples in plasma for stability studies.

Figure 10: Typical chromatogram for short term samples in plasma for stability studies.

Figure 11: Typical chromatogram for long term samples in plasma for stability studies.

To optimize the mobile phase, various proportions of sodium phosphate buffer (pH 4.0) with acetonitrile (HPLC Grade) were tested. The use of sodium phosphate buffer (pH 4.0) and acetonitrile (HPLC Grade) in the ratio of 70:30 v/v resulted in peak with good shapes and resolution. A flow rate of 1.0 mL /min was found to be optimum in the 0.4-1.5 mL/min range resulting in short retention time, baseline stability and minimum noise.

The LLOQ obtained for metformin and alogliptin by the proposed method in plasma was 5.936 and 1.983 μg/mL respectively. The retention times obtained for metformin and alogliptin in plasma were 2.734 and 4.451 respectively. Quantitative linearity of drugs in plasma was obeyed in the concentration range of 300-700 μg/mL for metformin and 7.5-17.5 μg/mL for alogliptin respectively. The relevant regression equations were y=1192.x-38173 for metformin (r²=0.997) and y=2802x-1113 for alogliptin (r²=0.998) (where y is the peak area and x is the concentration of metformin and alogliptin (μg/mL)). The intra-day and inter-day drugs variations in plasma by the proposed method showed percentage relative standard deviation were less than 2%, indicating that the method is precise. The corresponding mean recoveries of the drugs in plasma were 98.40-100.17%. This reveals that the method is quite accurate. The percentage relative standard deviation obtained for the drugs spiked in plasma for stability studies were less than 2%.

The proposed HPLC method was found to be simple, precise, accurate and sensitive for the simultaneous determination of metformin and alogliptin. The method was validated as per ICH guidelines and all the parameters met within the acceptance criteria. Applicability of this method for simultaneous estimation of metformin and alogliptin in plasma was confirmed.

The authors are thankful to M/s Pharma Train, Hyderabad for providing reference samples of metformin and alogliptin and processed plasma.