Figure 1. Structure of Vildagliptin

Pioglitazone is a member of the class of thiazolidenediones that is 1,3-thiazolidine-2,4-dione substituted by a benzyl group at position 5 which in turn is substituted by a 2-(5-ethylpyridin-2-yl) ethoxy group at position 4 of the phenyl ring. It shows hypoglycemic activity. It has a role as an insulin-sensitizing drug, an EC 2.7.1.33 (pantothenate kinase) inhibitor and a xenobiotic. It is a member of thiazolidenediones, aromatic ether and a member of pyridines. [5] Pioglitazone triggers the nuclear peroxisome proliferator activated receptor-? (PPAR-?), which assist to the increased transcription of various proteins regulating glucose and lipid metabolism. [6,7] These proteins escalates the post-receptor actions of insulin in the liver and peripheral tissues, which assist to improved glycemic control with no increase in the endogenous secretion of insulin. [8, 9] The drug has been well tolerated by adult patients of all ages in clinical studies. Oedema has been reported with mono therapy, and pooled data have shown hypoglycemia in 2 to 15% of patients after the addition of pioglitazone to sulphonylurea or insulin treatment. There have been no reports of hepatotoxicity. [10]

Pioglitazone is an orally administered insulin sensitizing thiazolidinedione agent that has been developed for the treatment of type 2 diabetes mellitus. [11]

       
           
       
    Figure 2 Structure of Pioglitazone

MATERIALS & METHODS:

Chemicals and reagents:

 Dalton Pharma Chem, located in Vadodara, Gujarat, India, generously supplied us with a gift sample of Vildagliptin and Pioglitazone as our reference standard throughout the course of our study. Loba Chemie. Pvt. Ltd., Mumbai, India supplied all other solvents, chemicals, and excipients (specificity) made used in this research.

UV Instrumentation:

For the experiment, a Shimadzu UV visible spectrophotometer (double beam) with a paired quartz cell having a 1 cm path length (UV-1800, UV Probe, Shimadzu Corporation, Kyoto Japan) was utilized. Weighing was carried out using the Digital electronic balance Citizen & Contact (CY 220 & CY 223).

Method Parameters:

Diluent: Water : Acetonitrile (50: 50%, v/v)

Wavelength: ?1 = 210 nm; ?2 = 226 nm

Standard Preparation:

Vildagliptin Standard Stock Solution-I (VSSS-I):

Initially Prepare a Standard Stock Solution (VSSS-I) of by adding  5 mg of Vildagliptin in 10 ml volumetric flask & add 5 ml diluent, mix for 2 minutes and make the volume to 10 ml with diluent. (Conc. of Vildagliptin = 500µg/ml).

Pioglitazone Standard Stock Solution-II (PSSS-II):

Then prepare a Standard Stock Solution (PSSS-II) of Pioglitazone by adding 15 mg in 10 ml volumetric flask & add 5 ml diluent, mix for 2 minutes and make the volume to 10 ml with diluent. (Conc. of Pioglitazone = 1500 µg/ml). Then add 1.0 ml of VSSS-I & 0.1 ml PSSS-I in 10 ml volumetric flask and add 5 ml diluent and vortex and make up the volume with diluent. (Conc. of Vildagliptin= 50µg/ml &Pioglitazone = 15µg/ml).

Selection of Wavelength:

50µg/ml of VDG Working Standard and 15µg/ml of PIO Working Standard were scanned in the UV range of 190-400 nm. Both the spectrum was recorded. From the spectra wavelengths 210 nm (?max of VDG) and 226 nm (?max of PIO) were selected for analysis of both drugs using simultaneous method. (?1-210 nm and ?2-226 nm). The absorbance at ?1 and ?2 was measured and the concentration was calculated using following formula;

Cx=  (A2ay1-A1ay2)/(ax2ay1-ax1ay2)

Cy=  (A1ax2-A2ax1)/(ax2ay1-ax1ay2)

Where,

Cx and Cy are the concentrations of Vildagliptin and Pioglitazone, respectively,

A1 and A2 are the absorbances of sample at ?1 and ?2, respectively,

ax1 and ax2 are the absorptivity of Vildagliptin at ?1 and ?2, respectively,

ay1 and ay2 are the absorptivity of Pioglitazone at ?1 and ?2, respectively.

Validation:-

Linearity:

1. 5 samples of varying concentrations ranging from 80% to 120% were made.
2. The concentrations are given belo

Table 1: Concentration for linearity Study.

The sample preparations are given as below;

X ml of VSSS-I and Y ml of PSSS-II was diluted to 10 ml.

LOD/ LOQ:

Can be calculated by using AVONA Technique.

LOD=  (3.3× Std Error of Intercept)/(Coefficient of X variable 1)

LOD=  (10× Std Error of Intercept)/(Coefficient of X variable 1)

c. Repeatability :

A single sample was prepared as described and 6 Measurements were made from same sample; checked for RSD.

d. Accuracy:

1. Samples were made of 80%, 100% and 120% concentration as per Table 1.
2. Samples were measured in triplicate to calculate % RSD.
3. % recovery was also calculated.

e. Intra- & Inter-day Precision:

• The working standard and drug product samples were freshly prepared and analysed in morning and evening for Intra-day precision.
• The same working standard and drug product were used for analysis on 2nd day for inter-day precision.
• % RSD for Assay was calculated for the confirmation of precision.

RESULTS AND DISCUSSION:

Selection of analytical wavelength:

Wavelength of maximum absorption ?max was determined by scanning 50µg/ml of VDG Working Standard and 15µg/ml of PIO by UV-Visible double beam spectrophotometer from 200 to 400 nm using diluent as blank (?1-210 nm and ?2-226 nm).

       
           
       
    Figure 3: Vildagliptin showed ?max at 210 nm

       
           
       
    Figure 4: Pioglitazone shows ?max at 226 nm.

Procedure for Analysis of Tablet Formulation:

Then add 1.0 ml of VSSS-I & 0.1 ml PSSS-I in 10 ml volumetric flask and add 5 ml diluent and vortex and make up the volume with diluent. (Conc. of Vildagliptin = 50µg/ml & Pioglitazone = 15µg/ml).

The method was validated as per the ICH Q2 (R1) guidelines

Table 2: Analysis of Tablet Formulation by proposed methods:

Validation:-

The  proposed  method  was  validated  according  to  ICH  guidelines for  validation  of analytical procedures in order to determine the linearity, Range, LOD and LOQ. precision and accuracy. [12-15]

1. Linearity:

Its ability (with in a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. The calibration curve was constructed between concentration verses absorbance. Different concentrations of Vildagliptin& Pioglitazone varying concentrations ranging from 40 to 60 µg/ml and 12-18 ?g/ml were made. The sample preparations are given as below; X ml of Montelukastand Y ml of Fexofenadine was diluted to 10 ml.

Table 3:  Different concentration of Vildagliptin.

Figure 5: Linearity of Vildagliptin

Table 4:  Different concentration of Pioglitazone.

Figure 6: Linearity of Vildagliptin

The proposed method was found to be linear in the range of 40-60 and 12-18 ?g/ml with correlation coefficient 0.999 and 0.999 for Vildagliptin & Pioglitazone respectively. The calibration curve was constructed between concentration verses absorbance. It is shown into the Figure 5 and 6 resp. and result of linearity study shown in Table 3, 4.

2. Range:

40-60 and 12-18 ?g/ml for Vildagliptin& Pioglitazone.

3. Limit of detection (LOD) and Limit of quantification (LOQ):

The standard deviation of y-intercept of regression line were determined and substituted in the following equation for the determination of detection of limit and quantification limits.

Detection limit= 3.3 ?/s

Quantification limit= 10 ?/s

Where, ? is the standard deviation of y-intercept of regression line and s is the slope of the calibration curve. The limit of detection (LOD) and limit of quantification (LOQ) data are given in Table 5.

The LOD and LOQ for Vildagliptin& Pioglitazone were determined according to ICH guideline

LOD = 3.3 ? / S LOQ = 10 ? / S

Where,

? = Standard deviation of the y intercept of calibration curves

S = Slope of the calibration curve

Table 5: LOD and LOQ of Vildagliptin & Pioglitazone.

The LOD and LOQ of Vildagliptin were discovered as 3.08 and 9.323 ?g/ml, individually, whereas for Pioglitazone was 0.68 and 2.06 ?g/ml, separately.

Limit of Detection and Limit of Quantitation was calculated on the basis of Slope and Standard deviation of response.

4. Repeatability

??????? Table 5: Repeatability of Vildagliptin & Pioglitazone

5. Precision:

 The precision of an analytical method is expressed as % RSD of a series of measurements which should be less than 2 %.( Table 6).

Table 6:  Intraday and Interday precision for Vildagliptin & Pioglitazone

The % RSD (< 2>

6. % Recovery (Accuracy):

Accuracy of the methods was determined at three different concentration levels i.e.80%, 100% and 120% in triplicate for each drug as per ICH guidelines. Method accuracy was evaluated as the percentage of recovery of known amounts of Vildagliptin & Pioglitazone. It is performed at spike concentration that was 80%, 100% and 120%.

Table 7. % Recovery data of the Vildagliptin.

Table 8. % Recovery data of the Pioglitazone.

The proposed procedures’ accuracy was determined using the standard addition method for recovering analyte’s. For each medicine, recovery rates in the experiments ranged from 98.88 % to 100.42 % for Vildagliptin and 98.74 to 101.37 % for Pioglitazone, proving the efficacy of the established protocols (Table 7, 8) Results of the analysis of pharmaceutical formulations reveal that the proposed methods are suitable for their simultaneous determination with virtually no interference of usual additive present in pharmaceutical formulations. Hence, the above methods can be applied successfully for simultaneous estimation of Vildagliptin and Pioglitazone in marketed formulations.