Method Development And Method Validation Of Oxycodone And Acetaminophen BY RP-HPLC

1.0       Introduction to HPLC:

High Performance Liquid Chromatography (HPLC) was derived from the classical column chromatography and, is one of the most important tools of analytical chemistry today.1In 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.2 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.3

1.1       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.5

Steps involved in Method development are. 4,5

         Understanding the Physicochemical properties of drug molecule.

         Selection of chromatographic conditions.

         Developing the approach of analysis.

         Sample preparation

         Method optimization

         Method validation.

1.2       Method Validation:

Validation of an analytical method is the process by which it is established by laboratory studies, that the performance characteristics of the method meet the requirements for the intended analytical application. Validation is required for any new or amended method to ensure that it is capable of giving reproducible and reliable results, when used by different operators employing the same equipment in the same or different laboratories. The type of validation program required depends entirely on the particular method and its proposed Applications.6Results from method .Validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice. Use of equipment that is within specification, working correctly and adequately calibrated is fundamental to the method validation process. Analytical methods need to be validated or revalidated.7

         Before their introduction into routine use;

         Whenever the conditions change for which the method has been validated

         Whenever the method is changed

Typical parameters recommended by FDA, USP, and ICH are as follow.7,8

1.         System suitability

2.         Precision

3.         Accuracy (Recovery)

4.         Linearity

5.         Robustness

6.         Limit of Detection (LOD)

7.         Limit of Quantification (LOQ)

2.         MATERIALS

Tools-Instruments:

•           HPLC – Waters Model NO.2690/5 Compact System consisting of panel with Inertsil-C18 ODS.

•           Electronic equilibrium (SARTORIOUS)

•           Sonicator (CLEAN FAST)

Chemical Substances:

•           Methanol grade HPLC.

•           HPLC Class buffer (KH2PO4).

Raw Stock:

Standards in dealing with Oxycodone and Acetaminophen.

2.1 METHOD FOR HPLC DEVELOPEMENT

Cellular phase: Methanol and Acetonitral were degassed at a 85:15 V/V ratio.

Preparation of a stock solution:

Reference remedy:

In order to prepare the solution, two volumetric flasks with a volume of 100.0 mL each were filled with 125.0 mg of Acetaminophen and 120.0 mg of precisely weighed Oxycodonethen sonicated for 20 minutes. Take 10.0 mL of each of the solutions and place them in a 100.0 mL volumetric flask. Next, add mobile phase, and after 10 minutes, sonicate the mixture.

The creation of a Standard Working Solution:

Along with the previously mentioned Oxycodone and Acetaminophen, For each medically, stock solutions varying in ranging from 20 to 80 ppm were made, sonicated, and filtered through a 0.45 membrane.

Optimised Chromatography Condition

 

 

Fig 1: Conventional Chromatogram

 

 

Fig: 2- Chromatogram compatible with devices (Standard 1)

 

 

Fig-3:Accuracy Chromatograms (50 percent)

 

 

Figure 4: Accuracy Chromatograms (100 per cent)

 

 

Figure -5: For Accuracy Chromatograms (150 per cent)

 

 

Fig: 6(a) Linearity Plot of Oxycodone (Concentration Vs response)

 

 

Fig: 6(b) Acetaminophen linearity plot (Concentration Vs response)

 

 

Fig-7: Robust Chromatograms

CONCLUSION

The analytical approach was developed through analysis of various parameters. First, maximum absorbance was observed at 254 nm for Oxycodone and 284 nm for Acetaminophen . Typical wavelength would be 247 nm, and the purity of peaks was excellent. The volume of injection was chosen as 20μl which gave a good peak area. The column used for analysis was Inertsil C18, a strong peak shape chosen by ODS. It has been found that ambient temperature suits the nature of the drug solution. Due to good peak area, adequate retention time and good resolution the flow rate was set at 1.0ml / min. Different mobile phase ratios were tested, mobile phase with Methanol and Acetonitrile (85:15) ratio was set due to good symmetrical peaks and good resolution. For the proposed analysis, instead, this mobile process was used.

The current recovery was found to be linear and precise over the same range as 98.0-101.50. Precision of both the device and the procedure has been found to be reliable and within limits. The detection limit for Acetaminophen was 0.25 Oxycodone, and 0.34. The study of linearity was, coefficient of correlation and curve fitting was found to be. For both the drugs, the analytical approach observed linearity over the 20-70ppm range of the target concentration. The analytical has passed tests of both robustness and robustness. Relative standard deviation was very satisfactory in both occasions.

REFERENCES

1. V. Gupta, A.D. K. Jain, N.S. Gill, K. Gupta, Development and validation of HPLC method - a review , Int. Res J Pharm. App Sci., (2012);2(4) 17-25
2. Y. Kazakevich, R. Lobrutto, HPLC for Pharmaceutical Scientists, John Wiley & Sons, New Jersey, 2007.
3. S. Ahuja, H. Rasmussen, Development for Pharmaceuticals,Separation Science and Technology, Elsevier, New York [2007] Vol.8
4. M.S. Charde, A.S. Welankiwar, J. Kumar, Method development by liquid chromatography with validation, International Journal of Pharmaceutical Chemistry, (2014);04(02): 57-61.
5. S. Sood, R. Bala, N.S. Gill, Method development and validation using HPLC technique – A review, Journal of Drug Discovery and Therapeutics, 2014; 2(22): 18-24.
6. N.Toomula, A. Kumar, S.D.Kumar, V.S. Bheemidi, Development and Validation of Analytical Methods for Pharmaceuticals, J Anal Bioanal Techniques. (2011); 2(5): 1-4.
7. T. Bhagyasree, N. Injeti, A. Azhakesan, U.M.V. Rao, A review on analytical method development and validation,International Journal of Pharmaceutical Research & Analysis, Vol (2014); 4(8): 444-448.
8. V. Kumar, R. Bharadwaj, G.G., S. Kumar, An Overview on HPLC Method Development, Optimization and Validation process for drug analysis, The Pharmaceutical and Chemical Journal,(2015); 2(2) ; 30-40