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. The development of new chemical entities (NCEs) is comprised of two major activities: drug discovery and drug development. The goal of the drug discovery program is to investigate a plethora of compounds employing fast screening approaches, leading to generation of lead compounds and then narrowing the selection through targeted synthesis and selective screening (lead optimization).
Over 25 years ago, Horvath and Melander, in their fundamental work , discussed the reason behind the explosive popularity of reversed-phase liquid chromatography (RPLC) for analytical separations. It was estimated that about 80–90% of all analytical separations were performed in RPLC mode, and the authors noted that “the variation of eluent composition alone extends both retention and selectivity in HPLC [high-performance liquid chromatography] over an extremely broad range.
High-performance liquid chromatography (HPLC) is a separation tool par excellence for the analysis of compounds of wide polarity. Since its inception approximately four decades ago, HPLC has revolutionized numerous disciplines of science and technology. Among the various modes of HPLC, reversed-phase and normal-phase chromatography (NPC) are employed most commonly in separation. Normal-phase chromatography was the ﬁrst liquid chromatography mode, discovered by M. S.
The process of analyte retention in high-performance liquid chromatography (HPLC) involves many different aspects of molecular behavior and interactions in condensed media in a dynamic interfacial system. Molecular diffusion in the eluent ﬂow with complex ﬂow dynamics in a bimodal porous space is only one of many complex processes responsible for broadening of the chromatographic zone.
The column is the only device in the high-performance liquid chromatography (HPLC) system which actually separates an injected mixture. Column packing materials are the “media” producing the separation, and properties of this media are of primary importance for successful separations.
The primary focus of this chapter in on general approaches and considerations toward development of high-performance liquid chromatography (HPLC) methods for separation of pharmaceutical compounds, which may be applied within the various functions in the drug development continuum. It is very important to understand the aim of analysis and the requirements for a particular method to be developed. The aim of analysis of each HPLC method may vary for each developmental area in the drug development process and speciﬁc examples are given in Section 8.2.
In modern high-performance liquid chromatography (HPLC), computers in a broad sense are used in every instrumental module and at every stage of analysis. Computers control the ﬂow rate, eluent composition, temperature, injection volume, and injection process. Detector output signal is converted from analog form into the digital representation to recognize the presence of peaks, and then at higher level of computer analysis a chromatogram is obtained. All these computer-based functions are performed in the background, and the chromatographer usually does not think about them.
Developing fast high-performance liquid chromatography (HPLC) methods can improve work efﬁciency during research, development, or production of a drug substance or a drug product. HPLC is a key technique in all of these areas. Until recently, analysis times of greater than 30 minutes were common. Modern pharmaceutical R&D, with its high-throughput screening, demands high-throughput methods to deal with the large number of samples. To reduce production cycle time, fast HPLC methods are essential for on-line or at-line process control and for rapid release testing.
Great efﬁciencies have been achieved in the drug discovery process as a result of technological advances in target identiﬁcation, high-throughput screening, high-throughput organic synthesis, just-in-time in vitro ADME (absorption, distribution, metabolism, and excretion), and early pharmacokinetic screening of drug leads. These advances, spanning target selection all the way through to clinical candidate selection, have placed greater and greater demands on the analytical community to develop robust high-throughput methods.
Analytical technology transfer and manufacturing is the mechanism by which knowledge acquired about a process for making a pharmaceutical active ingredient or dosage form during the clinical development phase is transferred from research and development to commercial scale-up operation or shared between internal groups or with third parties. Analytical technology transfer guarantees that laboratories can routinely execute tests, obtain acceptable results, and be able to accurately and independently judge the quality of commercial batches.
The most widely used analytical separation technique for the qualitative and quantitative determination of chemical mixtures in solution in the pharmaceutical industry is high-performance liquid chromatography (HPLC). However, conventional detectors used to monitor the separation, such as UV, refractive index, ﬂuorescence, and radioactive detectors, provide limited information on the molecular structure of the components of the mixture. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) are the primary analytical techniques that provide structural information on the analytes.
The discovery of a new drug is a challenging task that includes (a) identifica-
tion of a biochemical target for certain diseases and (b) screening of a large
number of compounds from libraries of compounds arising from synthetic
chemistry, combinatorial chemistry, and natural product isolation for lead gen-
eration. The lead compound is then optimized based on biological activity,
selectivity, pharmacokinetic property, and metabolism.
Some typical equilibration times for various column dimensions are shown
in Table 8-7; however, these should only be used as a guide. If complete equi-
libration is not achieved, early eluting components may show differences in
retention from run to run. An experiment could be run such that three dif-
ferent methods could be run with different equilibration times.
Size-exclusion chromatography (SEC) separates polymer molecules and biomolecules based on differences in their molecular size. The separation process in simpliﬁed form is based on the ability of sample molecules to penetrate inside the pores of packing material and is dependent on the relative size of analyte molecules and the respective pore size of the absorbent. The process also relies on the absence of any interactions with the packing material surface. Two types of SEC are usually distinguished: 1.
The method validation process is to conﬁrm that the method is suited for its intended purpose. Although the requirements of validation have been clearly documented by regulatory authorities [ICH, USP, and FDA], the approach to validation is varied and open to interpretation. Validation requirements differ during the development process of pharmaceuticals. The method validation methodologies in this chapter will focus on the method requirements for preliminary and full validation for both drug substance and drug product.
Choice of Stationary Phase
Ideally for a reversed-phase separations, the retention factors (k) for all com-
ponents in a sample should lie between 1 and 10 to achieve separation in a
reasonable time. For a given stationary phase the k of a particular component
can be controlled by changing the solvent composition of the mobile phase.
However, the impact of eluent composition will depend on the type of sta-
tionary phase and the nature of the components in the mixture.
Nitrogen Rule: If the nominal molecular weight of an analyte
appears to be an even mass number, the compound contains an even number
of nitrogen atoms (or no nitrogen atoms). On the other hand, if the nominal
molecular weight of an analyte appears to be an odd mass number, the com-
pound contains an odd number of nitrogen atoms.
Figure 4-43. Adsorption isotherms of alkylsulfates on Hypersil-ODS from methanol/water (20/80) with 0.02 M phosphate buffer at pH 6.0. (Reprinted from reference 119, with permission.)
Figure 4-44. Capacity factor of tyrosinamide versus concentrations of dodecyl sulfate (upper curve), decyl sulfate (middle curve), and octyl sulfate (lower curve). (Reprinted from reference 119, with permission.)
The modern drug discovery process, in general, involves the identiﬁcation of a biochemical target (usually protein target), screening of synthetic compounds or compound libraries from combinatorial chemistry/natural sources for a lead compound, and optimization of the lead compound (activity, selectivity, pharmacokinetics, etc.) for recommending a potential clinical candidate.
Chirality plays a major role in biological processes, and the enantiomers of a bioactive molecule often possess different biological effects. For example, all pharmacological activity may reside in one enantiomer of a molecule, or enantiomers may have identical qualitative and quantitative pharmacological activity. In some cases, enantiomers may have qualitatively similar pharmacological activity, but different quantitative potencies.