High Performance Liquid Chromatography (HPLC) was derived from the classical column chromatography and is one of the most important tools of analytical chemistry today. In the modern pharmaceutical industry, high-performance liquid chromatography (HPLC) is the major and integral analytical tool applied in all stages of drug discovery, development, and production. HPLC is the method of choice for checking peak purity of new chemical entities, monitoring reaction changes is in synthetic procedures or scale up, evaluating new formulations and carrying out quality control / assurance of the final drug products. The Goal of HPLC method is to try & separate, quantify the main drug, any reaction impurities, all available synthetic intermediates and any degradants. High Performance Liquid Chromatography is now one of the most powerful tools in analytical chemistry. It has the ability to separate, identify, and quantify the compounds that are present in any sample that can be dissolved in a liquid. HPLC is the most accurate analytical methods widely used for the quantitative as well as qualitative analysis of drug product and used for determining drug product stability. HPLC principle is the solution of sample is injected into a column of porous material (stationary phase) and liquid phase (mobile phase) is pumped at higher pressure through the column. The principle of separation followed is the adsorption of solute on stationary phase based on its affinity towards stationary phase.

The technique of HPLC has following features.

• High resolution
• Small diameter, Stainless steel, Glass column
• Rapid analysis
• Relatively higher mobile phase pressure
• Controlled flow rate of mobile phase

HPLC Method Development

Methods are developed for new products when no official methods are available. Alternate methods for existing (Non-Pharmacopoeial) products are to reduce the cost and time for better precision and ruggedness. When alternate method proposed is intended to replace the existing procedure comparative laboratory data including merit/demerits are made available. The goal of the HPLC-method is to try & separate, quantify the main active drug, any reaction impurities, all available synthetic inter-mediates and any degradants.

Steps involved in Method development are

• Understanding the Physicochemical properties of drug molecule
• Selection of chromatographic conditions
• Developing the approach of analysis
• Sample preparation
• Method optimization
• Method validation

MATERIALS AND METHODS

Instruments-Instruments:

• HPLC –Waters Model NO.2690/5 series Compact System Consisting Inertsil-C18 ODS column.
• UV spectrophotometer (Systronics)
• Electronic balance (SARTORIOUS)
• Sonicator( FAST CLEAN)

Substances containing chemicals:

• Methanol HPLC Grade.
• Buffer(KH2PO4)Hplc Grade.

EXPERIMENTAL:

1. Stock and standard Working solution:
2. Nebivolol is used as working standard in method development
3. Stock Solution Preparation :Take 100mg Asciminib working standard in 100ml V.F add  methanol sonicate it 30minets,(That is 1000ppm solution).
4. iii. Further Dilution (or) Trails Solution: Take 10ml of above solution in 100ml V.F add Methanol up to mark sonicate it 10minets(That 100ppm solution
5. iv.  Selection of wave length: Scan standard solution in UV spectrophotometer between 200 nm to 400 nm on spectrum mode, using diluents as a blank. Asciminib   shows ?max at 274nm.     

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5. Development of an HPLC method :       

The goal of this study was to improve the assay technique for simultaneous quantification of Asciminib on literature surveys. As a result, the trials detailed below show how the optimization was accomplished.

6. Method Validation:

System Suitability: 

A Standard solution was prepared by using Asciminib   working standard as per test method and was injected Five times into the HPLC system.

The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD from five replicate injections for Asciminib, retention times and peak areas.

Table-1: Data Of System Suitability

Solutions of standard and sample were prepared as per the test method are injected into chromatographic system.

Repeatability:

System precision:

Standard solution prepared as per test method and injected five times.

Method precision:

Prepared five sample preparations individually using single as per test method and injected each solution. % relative standard deviation of individual Nebivolol, from the five units should be not more than 2.0%. The assay of Nebivolol should be not less than 98% and not more than 102.0%.

Intermediate precision (analyst to analyst variability):

A study was conducted by two analysts as per test method. The individual assays of Nebivolol should be not less than 98% and not more than 102% and %RSD of assay should be NMT2.0% by both analysts.

A study of Accuracy was conducted. Drug Assay was performed in triplicate as per test method with equivalent amount of  Asciminib  into each volumetric flask for each spike level to get the concentration of  Asciminib  equivalent to 50%, 100%, and 150% of the labeled amount as per the test method. The average % recovery of Asciminib   was calculated.             

A Series of solutions are prepared using Asciminib  working standard at concentration levels from 20ppm to 70 ppm of target concentration .

LOD AND LOQ (LIMIT OF DETECTION AND LIMIT OF QUANTITATION):

From the linearity plot the LOD and LOQ are calculated:  

Table6: Data Of Linearity

System to system variability:

System to system variability study was conducted on different HPLC systems, under similar conditions at different times. Six samples were prepared and each was analyzed as per test method. Comparison of both the results obtained on two different HPLC systems, shows that the assay test method are rugged for System-to-system variability.

A study was conducted to determine the effect of variation in flow rate. Standard solution prepared as per the test method was injected into the HPLC system using flow rates, 1.0ml/min and 1.2ml/min. The system suitability parameters were evaluated and found to be within the limits for 1.0ml/min and 1.2ml/min flow. Asciminib  was resolved from all other peaks and the retention times were comparable with those obtained for mobile phase having flow rates 1.0ml/min.