Analytical Method Development in Pharmaceutical Research: Steps involved in HPLC Method Development
Hamid Khan*
J K College of Pharmacy, Bilaspur, CG, India-495001
*Corresponding Author E-mail: khanhamid770@gmail.com
ABSTRACT:
High performance liquid chromatography (HPLC) has proven to one of the major analytical technique used in the qualitative and quantitative analysis of drugs worldwide. Majority of drugs prescribed in official pharmacopoeias are being analyzed HPLC. Method development and validation is the essential part in the drug development programme in pharmaceutical industries and are associated with various steps such as pre-formulation, formulation, production, quality control and quality assurance and marketing of pharmaceutical products. Analytical method development is complicated process which takes time from few hours to months. This review paper covers the basic practical aspects of HPLC and gives guidance on how to develop HPLC analytical methods. This review provides the brief introduction, need and various steps involved in HPLC analytical method development.
KEY WORDS: HPLC, Chromatographic technique, Analytical Method Development, Steps in Method Development.
INTRODUCTION:
High Performance Liquid Chromatography (HPLC) method development and validation play important role in the drug discovery, development and manufacture of chemical and pharmaceutical products. HPLC is one of the most widely used analytical technique in pharmaceutical industry. Drug analysis reveals the identification characterization and determination of the drugs in mixtures like dosage forms and biological fluids.
During manufacturing process and drug development the main purpose of analytical methods is to provide information about potency (which can be directly related to the requirement of a known dose), impurity (related to safety profile of the drug), bioavailability (includes key drug characteristics such as crystal form, drug uniformity and drug release), stability (which indicates the degradation products), and effect of manufacturing parameters to ensure that the production of drug products is consistent [1-23]. HPLC utilises a liquid mobile phase to separate the components of a mixture through solid stationary phase. The components themselves are first dissolved in a solvent and then forced to flow (via the mobile phase) through a column (stationary phase) under high pressure. The mixture is resolved into its components within the column and the amount of resolution is dependent upon the interaction between the solute components and the column stationary phase (immobile packing within the column). The packing material of the column is the basic feature for the growth of this technique which directly responsible for the chromatographic separations. The interaction of the solute with the mobile and stationary phases can be selected through different choices of both solvent and stationary phases [24-34]. This review paper covers the basic practical aspects of HPLC and gives information and guidance on how to develop HPLC analytical methods. Various critical steps related to HPLC analytical method development and its optimization are also described in this paper [35-42]. A line diagram of various steps involved in HPLC method development is represented in Figure 1.
Figure 1: Steps involved in HPLC method development
Purpose of HPLC Method Development:
The reasons for the development of HPLC methods for drug analysis are: 1) When there is no official analytical method for analysis of drug or drug combination available in the pharmacopoeias. 2) When there is no analytical method for the existing drug in the literature due to patent regulations. 3) When there are no analytical methods for the formulation of the drug due to the interference caused by the formulation excipients. 4) Analytical methods for the quantitation of the analyte in biological fluids are found to be unavailable. 5) The existing analytical procedures may need costly reagents and solvents. It may also involve complicated extraction and separation procedures. When develop an HPLC method, the first step is always to consult the chromatographic literature to find out if anyone else has done the analysis, and how they did it. This will at least give an idea of the conditions that are needed, and may save one having to do a great deal of experimental work. When there are no authoritative methods are available, new methods are being developed for analysis of novel products. To analyze the existing either pharmacopoeial or non-pharmacopoeial products novel methods are developed to reduce the cost besides time for better precision, accuracy and sensitivity. These methods are optimized and validated through trial runs. Alternate methods are proposed and put into practice to replace the existing procedure in the comparative laboratory data with all available merits and demerits. Method development encompasses many stages and can take months to complete, depending on the complexity and goals of the method.
Steps Involved in HPLC Method Development:
Collection of Literature:
Analytical method development starts with literature survey where in various pharmacopeias like IP, USP, EP, BP, JP etc and chromatographic journals are referred to check the availability of suitable analytical methods. If any suitable method is found, it is still necessary to perform method development, optimization and validation to prove that the method can be successfully adapted for its intended use.
Sample Information:
The knowledge of nature and type of sample play an important role in the development of new HPLC method. It is necessary that analyst should have information that whether sample is polar or non-polar in nature. Ultimately this gives the idea of selection of stationay phase (Column packing material) for proper chromatographic separation of compounds. Pure drugs, their impurities and degradation products, excipients, finished dosage forms should be collected and the method should be designed based on the best resolution between the closely related compounds. The structures of impurities, starting material, intermediates and degradation products are compared with the structure of drug substances and arrive at the polarity whether they are less polar or more polar than the compound of interest.
Selection of Solvents:
Another essential parameter for HPLC method development is solubility of the compounds in which it is dissolved. The solvent should be chosen based on the solubility of drug substances, their impurities and degradation products. The solvent should be compatible with the mobile phase to get a better peak shape of analyte. Consider the purity of solvents and only use HPLC grade solvents. In normal phase HPLC systems, non-polar solvents such as hexane, diethyl ether, dichloromethane, iso-propyl alcohol, iso-octane etc are used whereas reversed phase HPLC requires polar solvents such as water, acetonitrile ethanol or methanol. The choice of mobile phase is governed by the physical properties of the solvent. Factors which are considered to be essential for selection of particular solvent are polarity, miscibility with other solvents, chemical inertness, and toxicity. The polarity index gives an indication of the ability of a solvent to elute a compound from the column.
Selection of Stationary Phase:
The selection of stationay phase in terms of column packing or bonded phase can be based on the polarity of molecule and its by-products. For normal phase HPLC, it contains the silanol groups (hydroxyl group). For reverse phase HPLC, a wide variety of columns are accessible covering a large range of polarity by cross-linking Si-OH groups with linear alkyl chains like C8, C18 and phenyl groups (-C6H6), nitrile groups (-CN), different embedded hybridized groups amino groups (-NH2) etc. The particles can be either spherical or irregular. Irregular particles include in general higher surface areas and higher carbon loads. Spherical particles confer higher efficiency, improved column stability, quick stabilization and lower back-pressure when compared to irregularly shaped particles. Particle size for HPLC/UPLC column packing denotes the average diameter of the packing particles. Particle size has an effect on the back-pressure and the separation efficiency of column. The column efficiency (performance) and column back-pressure are inversely proportional to the square of the particle diameter. A well packed column with 3μm packings produces almost twice the separation efficiency of a comparable 5μm column. However, the 3μm column will have about a three-fold higher back-pressure compared to the 5μm column when operated with the same mobile phase and at the same flow rate. A high surface area normally present high retention times (RT) and resolution (Rs) for separating complex and multi component samples. A packing material with a thin pore size will contain a big surface area and vice versa. Surface area is determined by pore size. Pore size and surface area are inversely related. The pore sizes of a packing material specify the average size of the pores within each particle. Generally, pore size of 150 A° or less is selected for samples with molecular weights less than 2000 and a pore size of 300 A or larger for samples with molecular weights greater than 2000. The carbon load is the quantity of bonded phase attached to the base material expressed as the percentage of carbon. High carbon loads usually offer greater resolution and greater retention times for hydrophobic samples. Low carbon loads shorten run times and often show dissimilar selectivity.
Selection of Mobile Phase:
The mobile phase selection is one of the critical parameter as it encourages the solute and the stationary phase interactions. An appropriate care must be taken while selecting the mobile phase like use of strong acids, strong bases and halide solutions should be avoided.
Selection of Buffer pH and Type of Buffer:
Buffers are usually employed in the mobile phase to obtain consistent chromatographic results. Buffers are employed to control the retention of ionic analytes. When the analyte is in ionic form, it usually attains polar in nature and spends shorter time on the stationary phase and elutes quickly. To control the selectivity of the ionic analytes, the buffer pH plays a significant role. In general, when buffer pH increases, the acidic analytes gets ionized and become more polar in nature and conversely, when buffer pH decreases, the basic analytes gets ionized. The pH of the mobile phase selected should be at least 1.0 pH units from the analyte pKa value. This confirms that the analytes are either as 100% ionized or 100% non-ionized and it helps in controlling peak shape and the run to run reproducibility. It is always use buffer in aqueous portion of the mobile phase and it increases the ruggedness of the method.
Selection of Buffer Concentration:
The concentration of the buffer in the mobile phase has the influence on retention time and peak shape of analytes in HPLC analysis. A concentration of buffer in the range of 5-100mM is usually sufficient for the most chromatographic separations. The concentration should be low enough to avoid the pumping problems due to the precipitation of buffer in HPLC.
Selection of Organic Additives:
The addition of mobile phase additives is usually to control the secondary interactions of the residual silanols on the stationary phase with the analyte. Triethylamine (TEA) or Diethylamine (DEA) are preferred in the mobile phase to control the peak tailing of basic analytes and acetic acid was preferred for acidic analyte.
Selection of Detector:
The selection of detector is based on the presence or absence of chromophores in the analyte. But, majority of pharmaceutical compounds exhibit UV spectra in the range of 200-400 nm. The most common detectors for HPLC system UV-Visible detector but PDA (Photodiode Array) detector is now a day widely used for identification of drug impurities and degradation products. The non-chromophoric compounds can be analyzed by using fluorescence, mass detectors etc. To confirm the peak purity, PDA detector is employed in modern HPLC/UPLC system. It collects the spectra at each point of the peak, starting from the peak elution to peak end and compares the spectra of peaks collected at all points versus the peak apex and thus confirms the homogeneity of the peak. The different components of HPLC system is shown in Figure 2.
Figure 2: Different components of HPLC system
CONCLUSION:
Planned way of method development with the aim of drug analysis is critical to success for fast and effective analytical method development. The general approach for the method development for separation of pharmaceutical compounds was discussed with emphasizing the knowledge of availability of literature on analytical methods for analysis of drugs, nature of sample, selection of suitable solvent, how to select and optimize mobile phase, how to select stationay phase and type of detector employed. Apart from this other parameter such as modifications in the mobile phase by adding buffers and organic additives, are essential to improve the chromatographic separation, resolution, sensitivity and selectivity. The knowledge of solubility and pKa of compound is most important prior to the HPLC method development. Through this the selection of mobile phase and type of column packing are selected and optimized. Other important part of HPLC method is detection of analytes, which is selected by having the information on chemical nature of the analytes. The various steps involved in HPLC method development are discussed and it is recommended that these steps should be followed to develop cost effective, sensitive, selective, precise and accurate HPLC analytical methods.
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Received on 20.10.2016 Accepted on 27.11.2016
© Asian Pharma Press All Right Reserved
Asian J. Pharm. Res. 2017; 7(3): 203-207.
DOI: 10.5958/2231-5691.2017.00031.4