Bioanalytical Method Development And Validation For The Simultaneous Estimation Of Remogliflozin Etabonate, Vildagliptin And Metformine In Tablet Dosage Form

Antidiabetic drugs are medicines developed to stabilise and control blood glucose levels amongst people with diabetes. Type 1 diabetes is a disease in which the body does not make enough insulin to control blood sugar levels. Type 1 diabetes was previously called insulin-dependent diabetes or juvenile diabetes. Diabetes mellitus is a group of metabolic diseases characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Metabolic abnormalities in carbohydrates, lipids, and proteins result from the importance of insulin as an anabolic hormone. Low levels of insulin to achieve adequate response and/or insulin resistance of target tissues, mainly skeletal muscles, adipose tissue, and to a lesser extent, liver, at the level of insulin receptors, signal transduction system, and/or effector enzymes or genes are responsible for these metabolic abnormalities. The severity of symptoms is due to the type and duration of diabetes. Some of the diabetes patients are asymptomatic especially those with type 2 diabetes during the early years of the disease, others with marked hyperglycemia and especially in children with absolute insulin deficiency may suffer from polyuria, polydipsia, polyphagia, weight loss, and blurred vision. Uncontrolled diabetes may lead to stupor, coma and if not treated death, due to ketoacidosis or rare from nonketotic hyperosmolar syndrome.(1,2,3) Vildagliptin (VGT) [(S)-1-[N-(3-hydroxy-1-adamantyl) glycyl] pyrrolidine-2-carbonitrile], Fig. 1,is a new oral anti-diabetic drug belonging to the class of dipeptidyl peptidase-4 inhibitor (reduces glucose-induced glucagon-like peptide 1 and gastric inhibitory polypeptide secretion) 3 and is used as mono therapy in adults with type 2 diabetes mellitus treatment especially in patients inadequately controlled by diet and exercise alone(4,5).

Metformin Hydrochloride (MTF) is chemically known as [1-carbamimidamido-N, N dimethylmethanimidamide] (Fig. 2) is an oral anti-diabetic drug in the class of biguanides. It is used as the first-line drug for noninsulin-dependent diabetes mellitus treatment 11. It works as improving glycemic control factor through decreasing hepatic glucose production, decreasing glucose absorption, and increasing the insulin-mediated uptake of glucose.

Therapeautic indications of metformin competent is indicated as second line treatment of type 2 diabetes mellitus adult patients, particularly overweight patients, who are unable to achieve sufficient glycaemic control at their maximally tolerated dose of oral metformin alone 12 . The mechanism through which metformin HCl decreases blood glucose and lipid concentrations is by activation of the enzyme AMP-activated protein kinase (AMK) and the Peutz-Jeghers protein, LKB1, to regulate AMPK 13. Remogliflozin Etabonate is chemically Ethyl [(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-[5- methyl-1-propan-2-yl-4-[(4-propan-2- yloxyphenyl)methyl]pyrazol-3-yl]oxyoxan-2- yl]methyl carbonate Fig.1.Vildagliptin Chemically (2S)-1-[2-[(3-hydroxy1adamantyl)amino] acetyl]pyrolidine-2- carbonitrile  Remogliflozin Etabonate reduces glucose concentration in type 2 diabetes by blocking renal glucose reabsorption as Vildagliptin prevents the degradation of GLP-1 and GIP, Which are incretion hormones that promote insulin secretion and control blood glucose levels.

       
           
       
    Fig. 3 Remogliflozin Etabonate

MATERIALS AND EQUIPMENTS

The drugs, chemicals, reagents, instruments and filters used during the experiment. Metformine Hydrochloride, Remogliflozin Etabonate and Vildagliptin.  API Aarti Drugs Limited, Mumbai Maharshtra, Laurus Labs Limited, Vishakhapatnam, Aandhra Prasdesh

 Instruments Used:                       

• Methanol (gradient grade)
• Ammonium Dihydrogen Phosphate Buffer
• Water (HPLC grade)
• Human plasma

EXPERIMENTAL WORK

 Optimization of chromatographic condition for the estimation of Metformin Hydrochloride, Vildagliptin and Remogliflozin

Solubility Studies:

As a first step of method development solubility of drugs was tasted in different solvents to obtain a suitable solvent which can be used for method development.

Selection of wavelength

By scanning between 200 and 400 nm, UV spectrum of 10 µg/ml Metformine Hydrochloride, Remogliflozin Etabonate And Vildagliptin in method was captured, a wavelength that provide a favorable reaction for the drug selection. 233 nm was chosen as the wavelength from the UV spectrum. At this Wavelength, drugs exhibited excellent absorption.

Selection of Mobile Phase

The prepare homogenous mixture of 850 ml HPLC grade methanol, 150 ml of buffer pH 3.0  shake well and through membrane filter paper. And sonicated the mobile phase for 5 min in sonicator. After trials Methanol: Buffer, 85:15 was found to be most satisfactory since it gave sharp peak with symmetry within limits and significant reproducible retention time.

Optimization of Chromatographic Condition:

The following chromatographic conditions were established by trial by error and were kept constant throughout the experimentation.

Preparation of stock solution

Weight accurately 125 mg MET, 25mg REMO and 12.5 mg VILDA and transferred into 100 ml volumetric flask then dissolved and diluted with diluent and make volume up to mark with help of diluent.

Preparation of Buffer

Ammonium Dihydrogen Phosphate Buffer: Weight Accurately 1.15 gm Ammonium Dihydrogen Orthophosphate dissolve with 900 ml HPLC grade water, add 1ml of Triethylamine shake well sonicate for 5min, adjust pH to 3.0 with diluted Orthophosphoric acid make up volume to 1000 ml with HPLC grade water.

Preparation of Standard solution

1. Metformine Hydrochloride standard Stock solution:

Accurately weighed quantity 125 mg of MET was dissolved in diluent and volume was made up to 100 ml mark (1250 µg/ml). The stock standard solution was diluted further with diluent to get final concentration of about 125µg/ml of MET.

2. Remogliflozin Etabonate standard stock solution:

Accurately weighed quantity 25 mg of REMO was dissolved in diluent and volume was made up to 100 ml mark (250 µg/ml). The stock standard solution was diluted further with diluent to get final concentration of about 25 µg/ml of REMO.

3.  Vildagliptin standard stock solution:

Accurately weighed quantity 12.5 mg of VILDA was dissolved in diluent and volume was made up to 100 ml mark (125 µg/ml). The stock standard solution was diluted further with diluent to get final concentration of about 12.5 µg/ml of VILD.

RESULT AND DISCUSSION

For method optimization various mobile phases were tried in different ratios, such as ACN: H2O: TFA (50: 50: 0.1) , MeOH: H2O (90: 10),  Buffer: ACN (50: 50) . All these mobile phases were unacceptable due to tailing, fronting and no sharpness in the peak. After various trials mobile phase consisting of Buffer: MeOH (85: 15) was selected which gave sharp peaks with no tailing and fronting. The chromatogram of standard was shown in fig.

Accuracy:

The concentration used were 80 %, 100 %, and 120% to analyte the recovery studies using the standard method. The procedure involved combining 0.8, 1.0, and 1.2 ml, of standard solution with 0.2 ml of solution having 10 µg/ml concentration.

       
           
       
    Fig. 4:- Typical chromatogram of standard solution.

       
           
       
    Table no 2 Accuracy data of MET by HPLC method

       
           
       
    Table no 3 Accuracy data of REMO by HPLC method

       
           
       
    Table no 4 Accuracy Data of VILDA by HPLC method

Linearity

1. The linearity for Metformin was determined in the range of 25-75µg/ml. The regression equation was found to be y = 27.33x -5.7783  R?2; = 0.9991. Data for calibration curve was shown in Table 3 and the calibration curve was shown in Fig

Table no 6 Calibration Standard Peak Area

Fig 5 Linearity and range of Metformin

The linearity for Remogliflozin etabonate was determined in the range of 25-75µg/ml. The regression equation was found to be y = 28.807x -25.635  R?2; = 0.999. Data for calibration curve was shown in Table 3 and the calibration curve was shown in Fig

Fig 6 Linearity and range of  Remogliflozin Etabonate

The linearity for Vildagliptin was determined in the range of 25-75µg/ml. The regression equation was found to be y = 17.22x -2.2601 R?2; = 0.999. Data for calibration curve was shown in Table 3 and the calibration curve was shown in Fig.

System Suitability parameter were shown to be within specified limits. Column efficiency (theoretical plates), resolution factor and peak asymmetry factor, tailing factor, LOQ and LOD are the system suitability parameter.

Limit of Detection (LOD)

The limit of detection is the lowest concentration of an analyte that can be detected in a sample but not necessary quantitated, under the given experimental conditions.

Limit of Quantification (LOQ)

It is the lowest concentration of analyte in a sample that can be accurately and precisely identified under the given experimental condition.

LOD and LOQ were determined using the following formulas

LOD = 3.3 × (SD) /S

LOQ = 10 × (SD) /S

Where, SD = Standard deviation

              S = Slope

For the parameter like flow rate, wavelength and the chosen solution was used for a robustness assessment. % RSD (NMT 2 ) should not be present in the variation. The percent assay should also fall between 98- 102 %

 %Assay determination of marketed formulation.

       
           
       
     Table no 18 Assay of Metformin Hydrochloride

       
           
       
    Table no 19 Assay of Remogliflozin Etabonate