Oct 4, 2014 -
World Journal of Pharmaceutical Research Sule et al.
World Journal of Pharmaceutical Research SJIF Impact Factor 5.045
Volume 3, Issue 9, 258-279.
Review Article
ISSN 2277– 7105
A PRACTICAL APPROACH TO RP HPLC ANALYTICAL METHOD DEVELOPMENT *Sunita Sule1, Sushma Ambadekar1, Deepak Nikam1, Ameet Sule2, Sudesh Bhure3 1 2
The Institute of Science, 15 Madame Cama Road, Mumbai 400032, India.
Presspart Manufacturing limited, Whitebirk Industrial Estate, Blackburn BB1 5RF, UK. 3
Cipla Ltd, LBS Marg, Vikhroli (East), Mumbai 400075, India. ABSTRACT
Article Received on 29 Sep 2014, Revised on 4 Oct 2014, Accepted on 08 Oct 2014
High performance liquid chromatography is one of the most widely used tools to identify and quantify potency in drug substances and drug products. Analytical method development and validation are two very critical processes performed before release of a method for use in
*Correspondence for
Quality Control department. This article focuses on stepwise practical
Author
approach towards developing a RP HPLC assay method. The various
Sunita Sule
contributing parameters and its effect on the performance of the RP
The Institute of Science, 15 Madame Cama Road, Mumbai 400032, India.
HPLC analytical method being developed are described simply, such that a new chromatographer is able to develop a method with the understanding of the RP HPLC method development process and its parameters.
KEY WORDS: Assay, Method development, Forced Degradation, Method Validation and High Performance Liquid Chromatography (HPLC). INTRODUCTION Despite the advances in technology, increased understanding of the analytical process and excellent softwares available for HPLC method development, there is an element of trial and error in HPLC method development. This can be reduced if not eliminated by proper planning. This article focuses on the process of developing a robust and stability indicative RP HPLC assay method for a pharmaceutical formulation by HPLC. A lab notebook or any other means to record the activities, output of literature and analytical findings is assigned for each method being developed. This document would later on be the base reference for writing the development report essential for transfer to QC lab and to sort any analytical issue which comes up later during validation or routine testing. Method development process is www.wjpr.net
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influenced by the nature of the analyte(s) and starts with defining the strategy generally as per the flow path shown in fig 1. Defining the Goal Understanding the Sample Literature survey Choice of method and Method input variables Method Development Method optimization and verification of robustness range Forced degradation studies Method validation Control Strategy Figure 1: Flow path for method development activity Defining the Goal Analytical method development can start by stating the purpose of the method. This information is derived from the quality document - Quality Target Product Profile. Analytical Target Profile (ATP) is developed for each of the attributes defined in the QTTP. The ATP for the test of assay would state the limit for the release for a quality control analytical method and the justification for the limit set. While defining the ATP some additional considerations can be requirements of fast analysis using low solvent as against max resolution of component as in case of test for impurities. In some cases the sample qty may be limited thus a highly sensitive method may be required. Another important activity while defining the ATP is the for the required system suitability criteria needs i.e. the resolution, theoretical plates, tailing factor, runtime. Many a times only tentative limits for system suitability criteria are stated initially and are finalized after the method validation is complete so that realistic limits can be set. Reference can be made to ICH Guidance e.g. ICH Q6 A and Q2 R1 Understanding the Sample Once the ATP is defined the next step would be to understand the sample. Describe and record the formulation details e.g. the active ingredient; single or multiple API, excipients and primary and secondary packaging if any. This is followed by studying the physico-
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chemical properties of the API (analyte), this information will help as an input for designing the analytical method development strategy.
Analyte characterization can be done by
conducting an organized literature search along with some simple lab tests and compiling the data for future reference. Some important analyte information and their contribution towards method development are listed in table 1. Table 1: Analyte Physicochemical Properties Sr.no 1
2
3
Information Contribution towards the RP HPLC method development Molecular Identification of the mode of HPLC testing i.e. a regular RP HPLC weight or Normal HPLC OR Molecular Weight separation technique. i) Identification of functional group - which dictates the choice of column chemistry and detector - whether UV/Vis, fluorescent, refractive index detector or ECD. Molecular ii) Identification of the nature of the molecule i.e. acidic, basic, structure neutral, stereo-isomers or Chiral molecule; which dictates the method input variables eg. stationary phase and operating pH iii) Knowledge of functional group will also help predicting the possible degradation route i) Selection of suitable diluent and mobile phase Solubility and solution stability
4
pKa value
5
Sample matrix
6
Sensitivity to heat
7
Sensitivity to light
ii) Sample solution concentration suitable for detection in the linear range of the detector iii) Solution stability would help in identify degradation pathway of the analyte. Choice of pH of the mobile phase for optimized separation. However if the pKa of the compound is beyond pKa value range () , Log P should be considered for selecting appropriate mode of chromatography The presence of excipients and their properties will contribute towards sample solution preparation; Generally techniques like filtration or centrifuging to remove insoluble excipient suffice but in some cases extraction or derivitization may be required. i) Sonication generates heat which could cause degradation of heat sensitive analytes in such cases alternate techniques for dissolving analyte needs to be evaluated ii) Restricted use of column temperature in the RP HPLC method being developed. Appropriate measures for sample preparation for light sensitive material eg. use of amber colored glassware, subdued light is advised
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Precautions to be taken while preparing sample solution
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Ensure that the analyte is soluble in the Diluent
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Concentration of the analyte solution should be within the linear range of the detector
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Based on the sample solution properties it is some time advisable to use guard columns.
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Literature Survey When developing an HPLC method, the first step is always to conduct a literature survey; copious information is generally available on physico chemical properties for the analyte of interest. Literature survey helps to ascertain whether the chromatographic separation has been previously performed and if so, under what conditions - this will save time doing unnecessary experimental work. Literature surveys can be conducted by referencing monographs prescribed in the pharmacopeias, chromatography articles and journals. Most of this data is now available online making the referencing process easy. Choice of Method and Method Input Variables Reverse phase chromatography is the first choice of separation for most regular samples due to its simplicity and high performance (selective) capabilities. The wide variety of equipment, columns, eluents and operational parameters involved makes high performance liquid chromatography (HPLC) method development seem complex, however it is this wide variety which helps make the analysis selective. This article specifically details the steps to be followed for developing a stability indicating RP HPLC analytical method using UV/ Vis detector. The input variables which affect the HPLC method performance are expressed in Fig 2 and are described briefly. Armed with the knowledge of sample, each of these variables can be studied to arrive at the configuration best suitable for the analyte of interest.
Figure 2: Input variables contributing towards the performance of a RP HPLC method Column
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Qualified and calibrated HPLC system should be used for method development activity to avoid system related error. Consideration should be given to qualification, training and expertise of the analyst to ensure efficient and effective planning and execution of the method development activity. Choice of column is critical to achieve desired separation. There is ample information on columns available in literature from the column manufacturers, where information of existing and new developed product is discussed in details and from other suitable scientific publications which can help selection of a suitable column. Choice of column is broadly split into choosing the right column chemistry and column dimensions. Effect of the column chemistry and column dimension on the separation is listed in table 2 and 3. Columns of similar chemistry and dimensions but different brands may behave differently as each manufacturer uses different substrate silica particle with different purity of silica which can influence separation of analytes. Table 2: Effect of Column chemistry on HPLC separation S. No.
Property
1
Particle shape of Silica substrates
2
Pore size and Surface area
3
Particle size
4
Bonded Phase
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Effect The base Silica particles are available as irregular, spherical and in fused state. Spherical particle shape has an advantage of reduced back pressure. Most interaction between the analyte and the bonded phase occur at the surface of the particle, Porous particle have higher surface area than non porous particles. Packing with higher surface area offer longer retention and higher resolution. As a rule of thumb base material with higher surface area should be chosen for complex samples requiring high resolution. Generally a pore size of 200A and less is suitable for samples with molecular weight 5000 Da or less and pore size of 300 A is suitable for sample with MW > 5000 Da ( eg Peptides). Selecting particle size it is generally a trade-off between efficiency and column pressure. Efficiency is higher with smaller particles because a) small particles have greater surface area and hence result in better separation and b) they pack together more densely than the larger particles there by reducing empty spaces where sample dispersion can occur but the tighter packing also produces higher pressure. A 3 micron column will have almost double the efficiency that with 5 microns keeping other conditions constant but at a higher column pressure. Columns with 1.8 micron size bonded phase are also available these are used to a lesser extent at present. Ideally for simple samples choose a 5 micron packing and for resolving complex, multi component samples lower particle size column can be used. There are a large number of bonded phase available to meet the
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5
Bonding type
6
Carbon load
7
Endcapping
8
Retention Factor: (or Capacity Factor)
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needs of various analytes Alkyl Phase: Straight chain alkyl stationary phase like C1,C2,C4, C6, C8 or C18 dominate hydrophobic interactions and are most suitable for hydrophobic compounds. C8 and C18 are the most commonly used bonded phase, C8 column may exhibit different selectivity due to increased base silica exposure and as they are less hydrophobic than the C18 columns. Modified Alkyl Phase: Highly polar compounds are difficult to retain, for such analytes modified Alkyl Phases e.g. embedded polar group or a polar side chain which can tolerate high % of aqueous proportion in mobile phase can be used. Bonded phase suitable for high pH is ideal for highly basic compounds which are difficult to retain and may require mobile phase with high buffer pH. Phenyl: Phenyl stationary phases favor pi-pi interactions and hence are excellent for compounds containing aromatic groups or unsaturated bonds. Cyano and fluorinated phase: Most suitable for strongly basic, nitrogen-containing and halogenated species. Amino and silica phases: Traditionally used for the normal phase separation of polar analytes. Chiral column: A wide variety of chiral stationary phases (CSPs) are available for choice based on the chemical grouping present in the chiral analyte. Size exclusion columns: These are suitable for separation of proteins based on hydrodynamic volume or in general terminology based on their molecular size. Bonded phase consist of hydrophobic moiety bonded to silica. These can be monomeric using single point attachment or polymeric using multipoint attachment to the base material. Polymeric bonding offers increased column stability and is suitable for separation of planar compounds from non planar compounds, typically positional isomers. Alkyl phase with high ratio of carbon : hetro atoms are suitable for analyzing neutral compound. Carbon load refers to the ligand density of alkyl chain on silica base material and is a good indicator of hydrophobic capacity of the stationary phase. Higher carbon load gives higher hydrophobic capacities, greater resolution and greater retention time for hydrophobic or non polar compounds. Whereas low carbon load results in fast elution of hydrophobic compounds, shorter analysis time and are suitable for simple sample mixtures and sample which require high water content for solubility. Endcapping is the process of bonding short hydrocarbon chains to free silinol remaining after the primary bonded phase has been added to the silica base. End capping helps in reducing peak tailing specially incase of polar compounds that interact with the exposed base silanols. The term Retention factor, k', is a means of measuring the retention of an analyte on a column. When retention factor for an Vol 3, Issue 9, 2014.
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analyte is less than one, it is considered that the analyte is not retained in the stationary phase. High retention factors (greater than 20) mean that elution takes a very long time. Ideally, the retention factor for an analyte should be between one and five. The retention factor for analyte A is defined as; k'A = t R - tM / tM t R = Retention time of peak of interest and tM = retention time of a non retained peak Table 3: Effect of Column Dimension on HPLC Separation S. No.
Property
1
Column length:
2
Internal diameter;
Effect Longer column give higher selectivity and higher plate counts as against smaller column in which the run time is proportionally small, equilibration is faster, back pressure is lower and sensitivity is higher. It Standard column lengths available are 250 mm, 150 mm, 100 mm, 75 mm, 50 mm and 30 mm. Smaller column diameter provides increase in sensitivity and good peak can be obtained with smaller sample size while wider bore facilitates higher sample load, a decrease in internal diameter from 4.6 to 4.0 can reduce about 25 % of solvent consumption but it also produces higher back pressure.
Mobile phase In RP HPLC the mobile phase is more polar than the stationary phase and the analyte partitions between the stationary phase and the mobile phase depending on its chemistry. The mobile phase pH and composition are important variables which controls the retention in a reversed-phase HPLC. Mobile phase typically consist of a mixture of aqueous phase and organic phase generally Acetonitrile or Methanol. The aqueous phase can be water or can contain additives like buffers, salts, surfactants, ion pair agents and reagents e.g. TFA . These additives serve to maintain a pH to control ionization of analyte, enhance retention by forming ion pairs, reduce interactions between analyte and silica surface, and maintain solubility of analyte or a combination of these functions. Characteristics of the components of a mobile phase are discussed briefly. Buffers Most of the samples contain one or more acidic or basic functional groups i.e. they contain ionizable analytes. Acids are almost 99% ionized above 2 pH units, or non-ionized below its pKa, while bases are ionized below and non ionized above their pKa. The non-ionized form is less polar and more hydrophobic thus more strongly retained in a reversed-phase system. Hence acids will be more retained at low pH and bases will be more retained at high pH. www.wjpr.net
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Selection of buffer is easy when the pKa value of the analytes is known; the mobile phase pH should not be very near the pKa of the analyte as minor variation in pH could result in large changes in retention, it should ideally be ±1.5 pH units away from analyte pKa. Another important consideration is that silica based columns should be operated between pH 2 to 8. Below pH 2 bonded phase loss due to hydrolysis may occur and above pH 8 silica tend to become soluble. Choice of buffer is typically governed by the desired pH. For the most effective buffering, a buffer should be used within ±1 pH unit of the buffer‘s pKa. Where ion pair agent is being used the buffer should be chosen such that it ensures that the analyte is completely in ionic state. Buffer should be chosen such that they are compatible with column packing and instrument thus strong acid, base and halides are not preferred, Citrate buffers corrode stainless steel. Concentration of the buffer should be generally between 10-50 mM. Increase in buffer concentration may improve peak shape to a certain extent but it might be at the cost of selectivity. Buffers chosen should be evaluated for absorbance in the UV region. A partial list of common buffers used and their useful range are listed in table Table No. 4: List of Buffer used in HPLC Buffer TFA Phosphate pK1 Phosphate pK2 Phosphate pK3 Citrate pk1 Citrate pk2 Citrate pk3 Carbonate pk1 Carbonate pk2 Ammonia TEA (0.1%)
pKa 0.3 2.1 7.2 12.3 3.1 4.7 6.4 6.1 10.3 9.2 10.8
pH range 1.5 – 2.5 1.1 – 3.1 6.2 – 8.2 11.3 – 13.3 2.1 – 4.1 3.7 – 5.7 5.4 – 7.4 5.1 – 7.1 9.3 – 11.3 8.2 – 10.2 9.8 – 11.8
UV cutoff (nm) 210
