High Performance Liquid Chromatography – A Validation View

Punam S. Desai*, Rajeshwar V. Chavan, Nikita B. Amane, Sanmati D. Shete, Archana R. Dhole

Rajarambapu College of Pharmacy, Kasegaon, Dist – Sangli, Maharashtra, India – 415404.

*Corresponding Author E-mail: punamdesai8@gmail.com

ABSTRACT:

High-performance liquid chromatography is an analytical separation technique used for most uses, because its reproductiveness, sensitivity, and the utility to differentiate non-volatile species makes the method ideal for accurate certified visualization. Chromatography is defined as a set of technologies that is used to separate the constituents of the mixture. This technique includes two steps stationary and mobile steps. The division of components is based on the difference between the splitting properties of the two layers. The division of components is based on the difference between the splitting properties of the two layers. The proposed method was to separate, validate and identify compounds, simple, accurate, and fast. Analytical method development and validation play an important role in the research and creation of pharmaceuticals. These methods are used to ensure the performance, accuracy, efficiencies and performance of drug products. Different types of detectors used to identify the peak during the development of the system. Pharmaceutical analytical chemistry deals with new analytical techniques and analytical chemists use chemistry, physics, biology, engineering, computer science, etc. to develop analytical methods. Different principles related to such interstate sciences should be considered. For example, analytical instruments like physicists developed mass spectrometer have found excellent applications in pharmaceutical analysis. High-performance liquid chromatography is an analytical separation technique used for most uses, because its reproductiveness, sensitivity, and the utility to differentiate non-volatile species make the method ideal for accurate certified visualization. Chromatography is defined as a set of technologies that is used to separate the constituents of the mixture. This technique includes two steps stationary and mobile steps. The division of components is based on the difference between the splitting properties of the two layers. The division of components is based on the difference between the splitting properties of the two layers. The proposed method was to separate, validate and identify compounds, simple, accurate, and fast. Analytical method development and validation play an important role in the research and creation of pharmaceuticals. These methods are used to ensure the performance, accuracy, efficiencies and performance of drug products. Different types of detectors used to identify the peak during the development of the system. Pharmaceutical analytical chemistry deals with new analytical techniques and analytical chemists use chemistry, physics, biology, engineering, computer science, etc. to develop analytical methods. Different principles related to such interstate sciences should be considered. For example, analytical instruments like physicists developed mass spectrometer have found excellent applications in pharmaceutical analysis.

KEYWORDS: high performance liquid chromatography. limit of detection, limit of quantification.

INTRODUCTION:

High Performance Liquid Chromatography which is also known as High Pressure Liquid Chromatography. This is a popular analytical technique used for the partitioning, identification and quantification of each component of the mixture. The HPLC column is an advanced technique for liquid chromatography. The solvent usually flows through a column with the help of gravity, but in HPLC technology, the solvent will be forced into high atmospheres in 400 atmospheres so that the sample can be divided into different constitutes by helping to differentiate between affinity¹.

Now a day reversed-phase chromatography is the most commonly used separation technique in HPLC due to its broad application range. It is estimated that 65% (preferably 90%) of all HPLC divisions fall in the opposite direction. This is because the simplicity, versatility and scope of the reverse phase method are able to handle the combination of different polarities and atomic mass².

The time of retardation depends on the nature of the analysis and the stability and design of the mobile phase. When a particular analyte speaks (ends at the end of the column) it is called retention time. In general solvents, methanol and Acetonitrile are usually mixed with any type of mixing of water or organic fluid. The selection of solvents, additives and gradient depends on the stage and the analyte³.

HPLC classified by:

I. Based on mode of seperation⁴:

Normal phase chromatography:

Stationary phase is polar (hydrophilic) and mobile face is non-polar (hydrophobic).

Reverse phase chromatography:

Stationary phase is non-polar (hydrophobic) and mobile phase is Polar (hydrophilic).

· Polar-Polar bonds and Non Polar-Non Polar bonds have more affinity than Polar-Non Polar bonds.

· Reverse phase chromatography is more commonly used as drugs are usually hydrophilic.

II. Based on Principle of Seperation^5,6

1.Ion-exchange chromatography-

Fig.1

2. Adsorption chromatography -

Fig .2

3. Ion pair chromatography:

It is a form of chromatography in which ions in solution can be “paired” or neutralized and separated as an ion pair on a reversed-phase column. Ion-pairing agents are usually ionic compounds that contain a hydrocarbon chain that imparts a certain hydrophobacity so that the ion pair can be retained on a reversed-phase column.

Method development⁷:

Purpose of method development:

Drug analysis is involves identifying descriptions and drugs in combination of dosage form and biological fluid. During the production process and during the development of the drug, the main objectives of analytical methods are power (which may be directly related to the requirements of a known dose), impurity, Drug preservation profiles), bioavailability (including crystal form, drug assuagement and drug-release characteristics), stability (which shows reduced products) and the effect of production parameters to make sure the product of the drug product is consistent.

The reasons for the development of novel methods of drug analysis are:

a. When there is no official drug or drug combination available in the pharmacopoeias.

b. When there is no decorous analytical process for the existing drug in the literature due to patent regulations.

c. When there are no analytical methods for the formulation of the drug due to the interference caused by formulation excipients.

d. Analytical methods for the quantitation of the analyte in biological fluids are found to be unavailable.

e. The existing analytical procedures may need costly reagents and solvents. It may also involve burdensome extraction and separation procedures.

Analytical method development and validation play an important role in the research and development of pharmaceuticals. These methods are used to ensure the performance, accuracy, efficiencies and performance of drug products. There are many components to consider developing methods. Initially aggregates information about the physical, chemical properties of analyte (PKA, Log P, and Solubility) and determines which mode of detection is appropriate. Analysis (i.e., proper wavelength in case of UV inspection) 3. Most analytical development efforts certify stability indicating HPLC-methods. The main purpose of the HPLC-method is to try to reduce the main active drug, any reaction impurity, all synthetic intermediates available and any distortion levels⁶.

Figure 1

Steps involved in HPLC method development:

Steps involved in Method development are⁸:

·       Understanding the physiochemical properties of drug molecule

·       Selection of chromatographic condition

·       Developing of approach of analysis

·       Sample preparation

·       Method optimization

· Method validation

1. Understanding the physicochemical properties of drug molecules:

Physicochemical properties of a drug molecule play an important role in method development. For Method development one has to study the physical properties like solubility, polarity, pKa and pH of the drug molecule. Polarity is a physical property of a compound. It helps an analyst, to decide the solvent and composition of the mobile phase⁹.

2. Selection of Chromatographic mode:

Chromatographic modes based on the analyte’s molecular chromatographic mode based on the analyte’s weight and polarity. All case studies will focus on reversed-phase chromatography (RPC), the most common mode for small organic molecules. Ionizable compounds (acids and bases) are often separated by RPC with buffered mobile phases (to keep the analyte’s in a non-ionized state) or with ion-pairing reagents¹⁰.

3. Selection of Column:

The heart of a HPLC system is the column. Changing a column will have the greatest effect on the resolution of analyte’s during method development. Choosing the best column for application requires consideration of stationary phase chemistry, retention capacity, particle size, and column dimensions. The three main components of an HPLC column are the hardware, the matrix, and the stationary phase¹¹.

4. Developing the approach for analysis:

While developing the analytical method on RP-HPLC the first step which is followed, the selections of various chromatographic parameters like selection of mobile phase, selection of column, selection of flow rate of mobile phase, selection of pH of mobile phase. All of these parameters are selected on the basis of trials and followed by considering the system suitability parameter¹².

5. Sample preparation:

The purpose of sample preparation is to create a processed sample that leads to better analytical results compared with the initial sample. The prepared sample should be an aliquot relatively free of interferences that is compatible with the HPLC method and that will not damage the column¹³.

6. Method optimization:

Most of the optimization of HPLC method development has been focused on the optimization of HPLC conditions. The mobile phase and stationary phase compositions need to be taken into account. Optimization of mobile phase parameters is always considered first as this is much easier and convenient than stationary phase optimization¹⁴.

Method Validation¹⁵

· Specificity

· Selectivity

· Precision

· Accuracy

· linearity

· Range

· limit of detection

· Limit of quantitation

· Robustness

· Ruggedness

Specificity:

Specificity is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present. Specification of the test method is determined by comparing the test results by analyzing the samples from placebo particles obtained through sample analysis, upgrade products, or sample analysis, without impurity, placebo components. Specificity can best be demonstrated by resolution between the analyte peak and the other closely eluting peak¹⁶.

Precision:

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. The precision of an analytical procedure is usually expressed as the standard deviation or relative standard deviation of series of measurements. Precision may be either the degree of reproducibility or of the repeatability of the analytical procedure under normal conditions. Intermediate precision (also known as ruggedness) expresses within laboratories variations, as on different days, or with different analysts or equipment within same laboratory¹⁶

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. lt is determined by applying the method to samples to which known amounts of analyst have been added. It may often be expressed as the recovery by the assay of known, added amounts of analyze¹⁶

Linearity:

Linearity of a method is its ability to obtain test results that are directly proportional to the sample concentration over a given range. For HPLC methods, the linear relationship between detector response (peak area and height) and sample concentration is determined. The relationship can be directly on drug substance by dilution of standard stock or by separate weighing of the sample components, using the proposed procedures. Linearity should be evaluated by the visual inspection of the plot of an analyte concentration or the work of the material ¹⁷

Limit of detection and limit of quantification¹⁸:

Limit of detection:

LOD is taken as the lowest concentration of an analyte in a sample that can be detected, but not necessarily quantified, under the stated conditions of the test.

Limit of quantification:

LOQ is the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated conditions of test some common methods for the estimation of detection and quantitation limit is,

· Visual definition.

· Calculation from the signal-to-noise ratio.

· Calculation from the standard deviation of the blank

· Calculation from the calibration line at low concentrations DL / QL =F × SD

Where,

F: Factor of 3.3 and 10 for DL and QL, respectively .

SD: Standard deviation of the blank, standard deviation of the ordinate intercept, or residual standard deviation of the linear regression.

b: Slope of the regression line .

Robustness and Ruggedness¹⁹:

Robustness:

The strength of the analytical process is the measurement of its capacity, which remains unimpressive, but provides an indication of its credibility during the deliberate difference between the criteria and the general use. Strength evaluation should be considered during development and depends on the type of study process. The paradigms should show the reliability of the analysis about the predictive differences.

Ruggedness:

Generally, laboratories from laboratories and analyzers and analysts measure the results of the internal reproduction test variation in the expected conditions. Analytical Method Runt is the reproducibility of test results obtained by analyzing similar samples in different samples. It should change Various laboratories, analysts, tools, agents, temperatures, time etc.

Instrumentation20, 21, 22:

1) Solvent delivery systems:

Reciprocating pumps are the most favored solvent delivery systems in micro column LC, because of their rapid flow equilibrium and stability, large column back pressure compensation abilities and good possibilities for micro-gradient delivery Minute flows in the low μL and nL ranges, for columns with I.D.s < 500 μm, cannot be readily provided by direct pumping. For such a small flow, the use of split-streaming techniques is optional.

Solvent reservoir contained glass container reservoir. Mobile phase solvent in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentration varied depending on the composition of the sample.

2) Sample injector system:

Injection volumes range between a few nL up to approximately 1μL for 1.0 mm I.D. columns Injection valves are the most popular injection systems in HPLC. The injection can be single or automatic injection system. Provide injection of the liquid sample within the range 0.1-100 ml of volume with high reproducibility and under high pressure upto 4000 psi.

3) Detector:

There are many ways to find when a substance is going through a column. Usually UV spectroscopy is attached, which searches for a specific mixture. In many organic compounds, UV light of various wavelengths is absorbed. Different types of detectors will depend on the amount of light absorbed on the number of specific compounds going from the detector as follows

Types of detector²³:

1. UV-visible detectors:

The UV-visible absorbance detector is the most common HPLC detector in use today since many compounds of interest absorb in the UV (or visible) region (from 190–600 nm). Sample concentration, output as absorbance, is determined by the fraction of light transmitted through the detector cell by Beer’s Law: A ¼ logðI0=IÞ ¼ Ebc

2. Light Scattering Detectors:

It used most non volatile analyte’s they Works well with gradient HPLC; Better sensitivity than Refractive detector. Some limitation of this detector Requires the use of volatile buffers, optimization; Limited dynamic range; Reproducibility of method.

CONCLUSION:

IT can be conclude from the entire review, HPLC is a unique, versatile, universal, fundamental tool and researchers have used it easily in terms of operation, availability and expense. In the current work, large and tablet formulations attempted to develop a simpler and faster HPLC method for routine analysis of drugs. For this purpose, the analytical column solvent selection, mobile phase composition, flow rate, and detector wavelength were studied. The developed method conditions are subjected for validation under ICH guidelines.

REFERENCE:

1. Mukthi Thammana A Review on High Performance Liquid Chromatography (HPLC), Research and Reviews: Journal of Pharmaceutical Analysis 2016; 5(2):22-28.

2. B. Prathapa, Akalanka Deyb, G.H. Srinivasa raoa, P. Johnsona and P. Arthanariswaranc A Review - Importance of RP-HPLC in Analytical Method Development, International Journal Of Novel Trends In Pharmaceutical Sciences 2013; 3(1): 15-23

3. Malviya R, Bansal V, Pal O.P. and Sharma P.K High Performance Liquid Chromatography: A SHORT REVIEW, Journal of Global Pharma Technology2010; 2(5): 22-26.

4. Ms Pallavi Nemgonda Patil HPLC method development - A Review, Journal of Pharmaceutical Research and Education, 2017, 1(2), 243-260.

5. A Review on Step-by-Step Analytical Method Validation, Panchumarthy Ravisankar 1IOSR, Journal of Pharmacy www.iosrphr.org Volume 5, Issue 10 (October 2015), PP. 07-19.

6. Vibha Guptaet al Development and validation of HPLC method – a review, International Research Journal of Pharmaceutical and Applied Sciences, 2012; 2(4):17-25.

7. Santosh Kumar, Bhardwaj, K. Dwivedi and D. D. Agarwal, A Review: HPLC Method Development and Validation International Journal of Analytical and Bioanalytical Chemistry 2015; 5(4): 76-81.

8. M.S. Charde, A.S. Welankiwar, J. Kumar, Method development by liquid chromatography with validation, International Journal of Pharmaceutical Chemistry, 04 (02) (2014) 57-61.

9. M.W. Dong, Modern Hplc for practicing scientists, John Wiley and Sons, New Jersey, 2006.

10. P.K. Singh, M. Pande, L.K. Singh, R.B. Tripathi, steps to be considered during method development and validation for analysis of residual solvents by gas chromatography, Int. Res J Pharm. App Sci., 3(5) (2013) 74-80.

11. M.S. Charde, A.S. Welankiwar, J. Kumar, Method development by liquid chromatography with validation, International Journal of Pharmaceutical Chemistry, 04 (02) (2014) 57-61].

12. N. Toomula, A. Kumar, S.D. Kumar, V.S. Bheemidi, Development and Validation of Analytical Methods for Pharmaceuticals, J Anal Bioanal Techniques. 2(5) (2011) 1-4.

13. K. Kardani, N. Gurav, B. Solanki, P. Patel, B. Patel, RP-HPLC Method Development and Validation of Gallic acid in Polyherbal Tablet Formulation, Journal of Applied Pharmaceutical Science. 3 (5) (2013) 37-42.

14. Vibha Gupta, Ajay Deep Kumar Jain, N. S. Gill1, Kapil Gupt Development and validation of HPLC method- a review, International Research Journal of Pharmaceutical and Applied Sciences 2012; 2(4): 17-25.

15. Lloyd R.S Ynder, Joseph J. Kirkland, Joseph L.Glajch, Practical HPLC method development,A Wiley –Inter science Publication JOHN Wiley and Sons,Inc. 1997 :2.

16. Anastasia Zotou. An overview of recent advances in HPLC instrumentation, Central European Journal of Chemistry 2012; 10(3): 554-569.

17. Sowjanya P and Subashini D and Lakshmi Rekha K analytical validation parameter Research and Reviews: Journal of Pharmaceutical Analysis 2015, ISSN: 2320-0812 p-ISSN: 2347-2340

18. Santosh Kumar Bhardwaj, K. Dwivedi and D. D. Agarwal A Review: HPLC Method Development and Validation, International Journal of Analytical and Bioanalytical Chemistry 2015; 5 (4): 76-81.

19. Mayuri A. Patil, Dr. S. T. Patil and Dr. S. P. Pawar a review on method development and validation of hplc method, world journal of Pharmacy and pharmaceutical sciences 2018; 7(4): 339-351.

20. Methods for the determination of limit of detection and limit of quantitation of the analytical methods, 2015, IP: 46.249.68.126.

21. G. Lavanya, M. Sunil, M.M. Eswarudu, M. Chinna Eswaraiah, K. Harisudha and B. Naga Spandana Analytical Method Validation: An Updated Review, International Journal Of Pharmaceutical Sciences And Research (2017): 0.59, SJR (2017): 0.137

22. Panchumarthy Ravisankar, Ch. Naga Navya, D. Pravallika, D. Navya Sri, A Review on Step-by-Step Analytical Method Validation 2015; 5(10):7-19

23. Michael Swartz HPLC Detectors: A brief review, Journal of Liquid Chromatography and Related Technologies 2010, 33: 1130–1150.