Development and Validation of UV Spectrophotometric Method for Estimation of Ursodeoxycholic acid in Bulk Formulation

 

Suraj J. Patil*, Sandeep R. Kane, Shrinivas K. Mohite, Chandrakant S. Magdum

Department of Chemistry, Rajarambapu College of Pharmacy, Kasegaon- 415404, Dist-Sangli, India.

*Corresponding Author E-mail: patilsuraj0212@gmail.com

 

ABSTRACT:

A rapid and economic UV spectrophotometric method developed by using a solvent methanol: acetonitrile (48:52) composed together to estimate the ursodeoxycholic acid content in bulk and pharmaceutical dosage formulation. Pre-determined study of λmax is 216nm it was proved linear range of 1.0-10.0µg/ml and exhibited good correlation coefficient (R2=0.992) and excellent mean recovery (98.00-100.05%). This method was successfully applied to the determination of λmax of ursodeoxycholic acid and results were in good agreement with label claims. The method was validated statistically and by recovery studies for linearity, precision, repeatability and reproducibility the obtained result proved that the method can be employed for the routine analysis of ursodexycholic acid in bulks formulation.

 

KEYWORDS: Ursodeoxycholic acid, UV Spectrophotometry, method validation.

 

 

 

INTRODUCTION:

The positive effects of ursodeoxycholic acid (UDCA) in primary biliary cirrhosis were the first firm proof that bile acids can in some way contribute to human injury1.There are many concerns that should be discussed in future regarding the hepatoprotective function of UDCA2. We do not know in particular how chronic cholestasis causes fibrosis in the liver and whether UDCA can prevent or counteract this cycle. Most cholestatic diseases have an immune pathophysiological basis Mechanisms can be divided into three groups for the beneficial effects of UDCA: hepatoprotective effect, endogenous bile acid and bile flow effect and immune modulation3. UV spectrophotometric approach used in raw substances as well as in manufactured dose formulations with a diluent consisting of methanol for the determination of ursodeoxycholic acid.

 

The system developed was standardized and tested in line with International Harmonization Conference (ICH) guidelines.

 

MATERIALS AND METHODS:

Apparatus:

The Jasco V-630 UV/Visible spectrophotometer where used in 1cm matched quartz cell absorbance measurement’s.

 

Objective:

UV spectrophotometric method for quantifying ursodeoxycholic acid should be developed and validated.

 

Materials:

About pharma PVT. Ltd kindly provided bulk formulation and other all chemical substance of analytical lobachem PVT. Ltd reagent grade unless otherwise specified all solution have been formulated using methanol and acetonitrile. Recently designed approaches have always been used.

 

Determination of wavelength of maximum absorption:

Standard stock solution use of diluents to prepare a regular ursodeoxycholic acid stock solution (10mg/ml) then dilute 1ml to 10ml with the same diluents to obtain the reference solution for ursodeoxycholic acid 10µg/ml.

 

Linearity and range:

For linearity analysis, five different stock solution aliquots (2, 4, 6, 8 and 10µg/ml) were used and the data obtained were used for the linearity calibration plot. The detection limit (LOD) and the quantification limit (LOQ) for the test were also determined.

 

Intra-day precision (repeatability) and inter-day precision study:

Ursodeoxycholic acid tablets have been finely powder and the 10mg/ml solution of sample stock has been prepared according to the same stock solution sequence. Five separate aliquots of 10ml were diluted to obtain 2,4,6,8 and 10µg/ml concentration .in the following days, this process was repeated.

 

Stability study:

The samples prepared for repeatability studies were stored at room temperature for 24 hours and tested for Short-term stability the following day.

 

Specificity in the presence of excipients:

The specificity test was conducted using only excipients granules spectra, blank and sample is wall-mounted furthermore, the specificity was determined in order to determine the sample solution had interacted with quantification of drugs by rapid degradation by heat (60°C) for 72 hr.

 

 

Table 1 intra-day and inter-day precision determined for three different concentration of ursodeoxycholic acid (n=3)

Concentration

(μg/ml Ursodeoxycholic acid)

Intra-day precision

Inter-day precision

Absorbance measured

(Mean± SD)

RSD (%)

Average potency (%)

Absorbance measured

(Mean± SD)

RSD (%)

Average potency (%)

2

0.37454±0.242337

64.70

97.08

0.3224±0.166784

51.73

97.10

4

0.38028±0.243318

63.98

97.45

0.37824±0.221119

58.45

97.50

6

0.38998±0.239023

61.29

97.85

0.37708±0.20423

54.16

97.90

 

Table 2 Short term stability determined by the proposed method (n=3)

Conc. declared (μg/ml)

Conc. found (mean +- SD μg/ml)

RSD (%)

Average potency (%)

2

2.7943±1.457471

52.15870189

97.56

4

2.958533±1.468868

49.64854

97.45

8

3.292433±1.567896

47.62119

97.63

 

 

Fig 1. UV spectra of ursodeoxycholic acid

 

 

 

RESULTS AND DISCUSSION:

Method development and optimization:

Ursodeoxycholic acid is almost in-soluble in aqueous medium and freely soluble in organic solvents like methanol, acetonitrile. During the development phase the use of few millilitre’s of methanol as the diluents resulted in preferable outcome UV analysis. The solvent was optimized to methanol. The pre-determined wavelength of maximum absorption (λmax) was 216nm.

 

Method validation5:

Linearity and range:

The calibration curve obtained was validated by its correlation coefficient. The absorbance of the sample in the range of 1.0-10.0 µg/ml was linear with correlation coefficient (R2) greater than 0.992 the LOD and LOQ were calculated as 0.072µg/ml and 0.21µg/ml respectively.

 

Intra-day and inter-day precision:

The intra-day and inter-day precision study (Table 1) of the developed method confirmed adequate sample stability and method reliability were all the RSD were ≤5 %.

 

Stability:

Stability study’s results were within the acceptance range and indicated the sample stability over 24h (short-term).

 

Specificity in the presence of excipients:

The specificity of the analytical method was proved by the spectra of degradation of product of sample solution with that of accuracy of sample.

 

CONCLUSION:

The results and the statistical parameters demonstrate that the proposed UV spectrophotometric method is simple, rapid, specific, accurate and precise. Therefore, this method can be used for the determination of ursodeoxycholic acid in bulk formulations without interference with commonly used excipients and related substances.

 

ACKNOWLEDGMENT:

The author wishes to thank Professor Dr. Sandeep Kane and also Vice-principal Dr. S.K. Mohite of Rajrambapu College of Pharmacy, Kasegaon for their inspiration and motivation, which together are the true formulas for writing the scientific manuscript.

 

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Received on 06.03.2020          Modified on 02.04.2020

Accepted on 28.04.2020   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2020; 10(3):155-157.

DOI: 10.5958/2231-5675.2020.00028.9