Comparative Study of Two Spectrophotometric Methods for Simultaneous Determination of Metformin Hydrochloride and Saxagliptin

Around the world, only 30% of people with diabetes keep their glycated hemoglobin (HbA1c) level below 7% (53 mmol/mol) which shows that effective treatment is lacking for most patients. More than 415 million people have been diagnosed with diabetes globally and almost 36% of them are in the Western Pacific region according to International Diabetes Federation in 2015. Roughly 193 million people have diabetes and don’t know it and it’s expected that 1 in 10 adults worldwide will be diagnosed with the disease by 2040 [1,2]. Various studies have found that a combination of medications early on helps people achieve their blood sugar goals [3]. It is also shown that greater control of blood sugar at the start of treatment can reduce the risk of serious complications and death, even in the long term. Saxagliptin, an oral anti-diabetic drug, belongs to the class of dipeptidyl peptidase-4 (DPP-4) inhibitors and is used in treating type 2 diabetes. On oral administration, the cyanopyrrolidine-based selective and competitive drug saxagliptin becomes less potent after being metabolized into an active mono-hydroxy derivative known as 5-hydroxy saxagliptin. Saxagliptin was given regulatory approval as a DPP-4 inhibitor by the FDA in 2009. This medicine is popular because it has a low dose and few chances of causing weight gain. To determine and validate saxagliptin by applying HPLC and RP-HPLC needs much time and are costly whereas UV spectrophotometric method involves lower cost and is much faster [4,5]. To treat diabetes, DPP-4 inhibitors such as saxagliptin assist in changing the action of the hormones called incretins. Incretins help bring down blood sugar by assisting the body in consuming more sugar and by having the liver output less sugar [6]. Metformin is the most commonly prescribed drug to handle type 2 diabetes, along with diet and exercise. When adjusting diet and lifestyle doesn’t work well to manage blood glucose, global standards suggest the use of metformin to patients with type 2 diabetes. Monotherapy might not be effective for a long time which often requires doctors to prescribe a combination of medicines together. Various oral treatments can be taken along with metformin, yet some people find their side effects problematic and often they do not help much. Sulfonylureas can make people feel hypoglycemic and they are also prone to weight gain, whereas α-glucosidase inhibitors may result in abdominal pain, diarrhea and discomfort from excess gas while thiazolidinediones are related to weight gain, heart problems, fluid retention and higher chances of fractures. That’s why investigators are eager to find new treatments and approaches that affect the disease. Saxagliptin is similar to metformin in that they both improve blood sugar control, so using both of them might enhance the treatment. In literature a large number of methods for estimation of metformin hydrochloride in drug products, either alone or in combination with other drugs have been reported [7-12]. Similarly, some methods are reported for saxagliptin either alone or in combination with other drugs [13-30]. However, very few analytical methods are available for the estimation of saxagliptin and metformin hydrochloride drugs in the combined dosage form.  Yet, only a handful of analytical techniques are available for quantitative estimation of saxagliptin and metformin hydrochloride drugs when given in a combined dose. UV spectrophotometer methods are favored over other analytical techniques due to their wide range of applications, ease of use, robustness, and simplicity. Therefore, it was thought valuable to come up with and examine some basic, accurate and precise UV methods aiming to determine saxagliptin and metformin HCl in tablets.

MATERIALS AND METHODS:

Chemicals and Reagents:

The reference samples of metformin (MET) and saxagliptin (SGT) were procured as gift samples from Yarrow Chemicals, Mumbai and from Morepen Labs limited, Parwanoo, Himachal Pradesh, India, respectively. The commercial formulation (Kombiglyze XR tablets containing 500 mg of metformin and 5 mg of saxagliptin) was purchased from the local market. Throughout the experiment, analytical-grade materials and distilled water was used.

Preparation of Standard Solutions of Metformin and Saxagliptin:

Stock solutions of both the drugs were made by weighing and transferring accurately 100 mg of each drug to the separate 100 mL volumetric flask having 20 mL of methanol in each flask. The flasks were swirled to dissolve the solid. Volume was made up to the mark with distilled water, which gave 1000 µg mL-1 of the drug. 10 mL of the above primary stock solutions were taken in 100 mL volumetric flask and volume was made up to the mark with distilled water to give secondary stock solutions of 100 µg mL-1. From the secondary stock solutions, aliquots were diluted with distilled water to obtain working standard solutions of 10 µg mL-1 of both the drugs.

Selection of Analytical Wavelength:

Simultaneous equation method (Method I):

Scanning of standard solutions of concentration 10 µg mL-1 of MET and SGT in distilled water was carried out between 200-400 nm by using distilled water as blank to find out the λmax¬ of both the drugs. ?max of SGT and MET was found to be 215 nm and 233 nm, respectively (Figure 1).

(b)

(a)

One of the main purposes of dual-wavelength method is to estimate the amount of a specific substance in a mixture by measuring the difference in absorbance values at two different points on the mixture’s spectra. This difference in absorbance values is directly proportional to the concentration of component studied and is independent on the concentration of other interfering component. To use the dual-wavelength method, two such wavelengths are selected where the interfering component gives the same absorbance whereas the interested component differed significantly in absorbance as a function of the its concentration [31]. As per spectrum, the absorption of MET was same at 223 and 238 nm so that these wavelengths were selected for estimation of SGT and absorption of SGT was same at 207 and 220 nm, hence these two wavelengths were selected for estimation of MET. According to the spectrum, there was no difference in the absorption of MET at 223 and 238 nm, so these two were chosen for determining SGT. Absorption of SGT was same at 207 and 220 nm and these wavelengths were chosen for MET.

Calibration Curves for SGT and MET:

Simultaneous equation method (Method I):

From the secondary stock solutions, the solutions of 10-30 µg mL^-1 for SGT and 2-10 µg mL^-1 for MET were prepared using distilled water. The corresponding absorbances were measured at 215 nm (λ_max of SGT) and at 233 nm (λ_max of MET). Calibration curves were plotted by taking concentration on x-axis and absorbance on y-axis.

Dual wavelength method (Method II):

Final concentrations of 10, 15, 20, 25, 30 µg mL^-1 were obtained after taking appropriate aliquots of SGT secondary stock solutions into different 100 mL volumetric flasks and diluting with distilled water up to the mark. The absorbance at both 223 nm and 238 nm was determined using distilled water as a blank and the results were used to make a graph of the absorbance differences against concentration. Also, the appropriate aliquots of secondary MET stock solution were diluted up to the proper amounts in different 100 mL volumetric flasks using distilled water to prepare 2, 4, 6, 8 and 10 µg mL^-1 MET solutions. The absorbance readings for the solutions were made at wavelengths 207 nm and 220 nm against distilled water as the blank and a plot between the difference in absorbance and the Concentration of MET was made.

Estimation of Metformin and Saxagliptin in Tablet Dosage Forms:

Ten tablets were ground up after weighing. The powder in an amount equivaqlent to 100 mg MET was weighed and added to a 100 mL volumetric flask containing 20 mL of methanol. The contents of the flask were sonicated for around 15 min. so that the drug was thoroughly dissolved and distilled water was added to the required volume. The solution was passed through a Whatman filter paper (No. l). Further, 4 mL of the filtrate was transferred into a 100 mL volumetric flask and volume was adjusted to the mark with distilled water. Then, 10 mL of the previous solution was transferred into a 100 mL volumetric flask and the distilled water was added until it reached the mark.

Simultaneous equation method:

The measurements of the absorbance of solution of the sample were done at both λ₁ and λ₂. The process of analysis was carried out 6 times using the tablet formulation. Using equations 1 and 2, the quantities of SGT (C_x) and MET (C_y) in the binary test solution were calculated.

Where, A₁ and A₂ are the absorbances at both the wavelengths

ax₁ = Absorptivity of SGT at λ₁

ax₂ = Absorptivity of SGT at λ₂

ay₁ = Absorptivity of MET at λ₁

ay₂ = Absorptivity of MET at λ₂

Dual wavelength method:

The sample solutions were measured at selected wavelengths against distilled water as blank and the concentration was obtained by extrapolating difference in absorbance on the working standard curve. Six times, the analysis process was repeated witht the test formulation.

Method Validation:

The above methods were validated for linearity, precision, accuracy, and specificity by following procedure.

Linearity:

Linearity solutions of 10-30 µg mL^-1 for SGT and 2-10 µg mL^-1 for MET from secondary standard stock solution were prepared using distilled water. In simultaneous equation method, the absorbances of all the prepared solutions were measured at λ₁ (i.e. λ_max of SGT) and also at λ₂ (i.e. λ_max of MET). In case of dual wavelength method, the absorbances were found at 223 nm and 238 nm for SGT and at 207 nm and 220 nm for MET. The absorbance differences were calculated for SGT and MET at the specified wavelengths.

Three measurements were done for every concentration and the linearity was demonstrated by the regression analysis.

Precision:

Aliquots of 2.0, 2.5 and 3.0 mL of secondary stock solution (100 µg mL^-1) of SGT were transferred to a different 10 mL volumetric flasks. Aliquots of 0.6 mL, 0.8 mL and 1 mL of MET secondary stock solution (100 μg mL^-1), respectively were then put into the corresponding 10 mL volumetric flasks. Then, the required concentration of 20, 25 and 30 μg mL^-1 of SGT and 6, 8 and 10 μg mL^-1 of MET was obtained by adding distilled water up to the mark. The intra-day and inter-day precision study of the proposed method were carried out by measuring corresponding responses three times on the same day and on three different days, respectively for the above solutions and the results were reported in terms of relative standard deviation (RSD). The repeatability studies were carried out by estimating the response for MET at 10 μg mL^-1 and for SGT at 30 μg mL^-1 6 times each and % RSD was reported.

Accuracy:

The accuracy of the method was determined by calculating recoveries of MET and SGT by method of standard additions. To the fixed amount of drug sample solution, a known quantity of standard drug was added and the percent recovery was found. The standard addition method was completed for levels of sample concentration at 50%, 100% and 150%. All the dilutions for standard and sample were prepared from their secondary stock solutions of 100 μg mL^-1. According to the proposed method, the experiments were repeated thrice at every level and the average was taken into account. For every level, the percentage recovery was determined.

Limit of detection (LOD) and Limit of quantitation (LOQ):

To determine LOD and LOQ, the analytes were measured and quantified six times.

RESULTS AND DISCUSSION:

Method Validation:

Linearity:

By examining six solutions with concentrations of SGT and MET ranging from 10 to 35 μg mL^-1 and 2 to 12 μg mL^-1, respectively, linearity of the proposed methods was assessed. For SGT and MET, good linearity was demonstrated over the investigated concentration range for method I (Figures 2 and 3) as well as for method II (Figure 4). Linearity results for both the drugs are shown in Table 1 (methods I and II). The correlation coefficients for both the methods were found to be closer to one for both the drugs and hence passed the linearity test.

Table 1. Linearity Results for SGT and MET

By choosing three concentrations from the linearity range and evaluating them on the same day for intra-day and three different days for inter-day, the precision of the proposed approach was assessed. The %RSD for both intra-day and inter-day was found to be less than 2 which is in accordance with the permissible range (Table 2 and 3). % RSD for repeatability was also found to be less than 2% for both the methods (Table 4). As a result, the suggested methods were found to be precise for estimating both drugs.

Table 3. Interday Precision Test of SGT and MET

Table 4. Repeatability Test of SGT and MET

The accuracy results are presented in Tables 5 and 6. Satisfactory recoveries ranging from 97.96 to 102.09 % for SGT and 96.41 to 101.56 % for MET were obtained by the proposed method I whereas using proposed method II, recoveries ranging from 96.93 to 102.21% for SGT and 96.02 to 101.44 % for MET were obtained. Such a high percentage recovery (96?103 %) indicates that the procedure is well suited for the routine analysis of these drugs in the combined mixture.

The standard deviation of the regression line was employed to measure the LOD and LOQ by the formulas as:

LOD =3.3 s/S and

LOQ =10s/S

Where,

s represents the standard deviation of the regression line and

S represents the calibrated curve's slope.

The LOD and LOQ of MET and SGT are given in Table 7.

Estimation of Metformin and Saxagliptin in tablet dosage form:

It is important to conduct quantitative testing to establish if a medicine’s amount is correct as mentioned because too high a dose may cause harmful effects and too little will not help the patient.

Method I:

If two or more drugs are combined in a single dosage form, the simultaneous equation or Vierordt’s method can be used for their estimation. After examining the spectra of SGT and MET, 215 nm and 233 nm were picked as optimum wavelengths since the both drugs showed absorbance at these absorbances. The absorptivities of the two drugs were measured at both the wavelength and are given in Table 8. The concentrations of both the drugs SGT and MET were found by using the two equations (1) and (2) as given in the materials and methods section.

Table 8. Absorbance and absorptivity values of SGT and MET at 215 and 233 nm

The projected method was successfully applied for quantitatively determining SGT and MET in the dosage form. The assay obtained was 98.90 and 99.82 % for SGT and MET, respectively and hence the proposed method can be used for the simultaneous determination of the two drugs in combined tablet formulation (Table 9). This method was successfully used by several researchers for simultaneous estimation of various drugs [32-34].

SGT was quantitative determined by measuring the change in absorbance at 226 nm compared with 238 nm because MET had equal absorbance at both wavelengths. The difference in absorbance between 226 nm and 238 increased with increase in concentration of SGT in the mixture. The process of quantifying MET was done by measuring the change in absorbance at 209 nm and 217 nm while the absorbance of SGT at those wavelengths was unchanged. The difference in absorbance between 209 nm and 217 nm increased with increase in concentration of MET in the mixture. The proposed method was successfully used to measure the quantity of SGT and MET in tablet formulation. The assay of SGT was found to be 100.80 and that of MET to be 99.18 % which confirmed the suitability of the method for estimating both drugs in combined tablet formulation (Table 9). The same method was followed by Patel and Maheshwari for the simultaneous estimation of torsemide and amiloride hydrochloride in their combined dosage form, Parmar and Patel in the simultaneous estimation of paracetamol and piroxicam and Abdelwahab et al. for the estimation of atorvastatin calcium and ezetimibe in their combined formulation [31,35,36].

Statistical Comparison of Method I and Method II by One Way ANOVA

A statistical analysis was conducted on the assay results to check the effect of the two different strategies used. The one-way ANOVA method was chosen to determine if the two methods were different in statistical significance. Both tests were set to use a significance level of p?0.05. Table 10 shows the results of one-way ANOVA and found that the developed methods showed very little difference between them [37].