Bioanalytical stability indicating method development and validation for the estimation of Lobeglitazone Sulphate in human plasma by using RP-HPLC Method

In pharmaceutical industry, there is a need for the invention of suitable novel analytical methods from time to time for testing the quality of bulk drugs, excipients and formulations. Method development and validation is an integral part of drug discovery and drug development. UV-visible spectroscopy and HPLC are the most popular techniques used for the identification and estimation of drugs with good accuracy and precision. Simultaneous method development is useful for analysis of combination of drugs. Bioanalytical method development is used for the estimation of drugs and their metabolites in various biological fluids like blood, urine, plasma, serum, saliva and cerebrospinal fluid. Worldwide, around 463 millions people are suffering from type-2 diabetes mellitus [International diabetes federation,2019] Anti-diabetic drugs are used for treating type-2 diabetes mellitus. There will be always a need for the development and validation of novel analytical methods for the estimation of drugs in bulk and dosage forms in order to deliver good quality and affordable medicines to patients Degradation studies of the drug substance include both appropriate solution and solid-state stress conditions (e.g., acid/base hydrolysis, oxidation, heat, humidity, and light exposure in accordance with ICH guidelines). Applied stress conditions should result in approximately 10–20 % degradation of the drug substance or represent a reasonable maximum condition achievable for the drug substance. In case, if no degradation is observed under the specified stress conditions, it is recommended to stop stress testing. A better knowledge of therapeutics and physicochemical and toxicological behaviour of drug and its pharmaceutical formulation can be gained from degradation study. This study allows a formulator in formulating more stable drug preparation by using a suitable solvent or vehicle or ingredients, which will prevent or retard the degradation. Further, it helps in deciding the storage conditions and routes of administration of various pharmaceutical dosage forms.

Ø  Reagents and chemicals:

•              Methanol (HPLC Grade)

•              HPLC grade water

•              Acetonitrile (HPLC Grade)

Methanol and Acetonitrile HPLC grade was purchased from Merck limited, Mumbai. HPLC grade water used generated using LABLINK water purification system. Pooled plasma was obtained as gift sample from Akshay Blood Bank, Pune.

Ø          Instruments:

1.            HPLC (UV):

•          Borwin- software (version 1.50)

•          Model PU 2080 Plus Intelligent HPLC pump

•          Rheodyne sample injection port with 20 μl loop

•          Rubitas C18 Column (250 mm  4.6mm, 5 µm particle size)

•          Jasco UV 2075 plus detector

•          UV-Visible Double beam spectrophotometer (Shimadzu Model 1780)

2.       Shimadzu (model ATX-224) Electronic weighing balance

3.       Sonicator: Prama solutions for laboratory

4.       LABLINK–XTRAPURE water purification system. Conductivity below 0.055 μS/cm

5.       Electronic pH meter

6.       Cold Centrifuge (BL 165)

7.       Deep Freezer (Make: Classic)

v  Bioanalytical Method Development

v  Preparation of standard stock solution of Lobeglitazone Sulfate

For standard stock solution accurately weighed 10 mg of Lobeglitazone Sulfate transferred to 10 ml volumetric flask and the volume was made up to 10 ml with methanol, to get standard stock solution of Lobeglitazone Sulfate (1000 μg/ml). Further dilutions were made with methanol to produce the stock solutions of 1, 2, 5, 10, 20, 30, 40, 50 μg/ml.

v  Selection of Analytical Wavelength

A solution of 10 µg/ml was prepared from standard stock solution of Lobeglitazone Sulfate (1000 μg/ml) and scanned over 200-400 nm in UV Spectrophotometer. The maximum absorbance was shown at 248 nm. Hence it was selected as analytical wavelength

A Bioanalytical method was developed for the estimation of lobeglitazone in Human Plasma by HPLC method and was validated. Lobeglitazone is an antidiabetic drug from the class called thiazolidinedione. The method was developed using Acetonitrile:  Methanol (60:40 v/v). The peaks obtained for the drug of interest by the present method was symmetrical in nature with acceptable tailing factor and from the plasma endogenous proteins by Protein precipitation Extraction. The retention time of lobeglitazone was shorter and proves that the method is rapid. All the analytical validation parameters were determined and found in the limit as per ICH guidelines, which indicates the validity of the method. The method was validated with respect to accuracy, precision, linearity and robustness. The results of linearity, intraday and interday precision study and capability of the extraction method were within the limits of Bioanalytical method development. The method was linear with a correlation coefficient of acceptable agreement, which is suitable for the estimation of lobeglitazone human plasma. The method developed can be used in therapeutic drug monitoring units, bioequivalence and bioavailability studies, pharmacokinetic and toxicology studies of lobeglitazone.