HPLC method development case studies

This chapter provides an overview of modern HPLC method development and discusses approaches for initial method development (column, detector, and mobile phase selection), method optimization to improve resolution, and emerging method development trends. The focus is on reversed-phase methods for quantitative analysis of small organic molecules since RPLC accounts for 60-80% of these applications. Several case studies on pharmaceutical impurity testing are presented to illustrate the method development process. For a detailed treatment of this subject and examples of other sample types, the reader is referred to the classic book on general HPLC method development by L. Snyder et al.1 and book chapters2,3 on pharmaceutical method development by H. Rasmussen et al. Other resources include computer-based training4 and training courses.5... 

Table 8.6 provides descriptions and estimated costs for a number of software and automated systems for HPLC method development and optimization. The reader is referred to some key references1 12 and the vendors websites for further details. Examples on Dry Lab simulation, the first and the mostly widely used software, are shown in the case studies (Sections 8.8.1-8.8.3) of this chapter. 

In this section we are going to look at some case studies to see how hplc experimental methods are developed. 1 am not going to give a long list of applications, because these are easy to find elsewhere, and sometimes do not make very interesting reading. Most textbooks on hplc have lists of applications, eg the book by Hamilton and Sewell (2nd Edn, Chapter 8), and applications can also be obtained from a number of journals (eg Analytical Chemistry annual reviews). 

The primary object of this book is to provide the HPLC practitioner with a handy guide to the use of HPLC for analyzing pharmaceutical compounds of interest. This means familiarizing the practitioner with the theory, instrumentation, regulations, and numerous applications of HPLC. This handbook provides practical guidelines using case studies on sample preparation, column or instrument selection, and summaries of best practices in method development and validation, as well as tricks... 

This case study illustrates the SP method development of an assay method for a controlled-release analgesic tablet with a single API. Certain considerations were taken into account. First, the analyte within the tablet matrice core had to be extracted quantitatively. Second, the analyte was diluted into a final solution that was compatible with the HPLC mobile phase. Third, short SP time was required (i.e., 30 min) to maximize productivity of the work scheme for processing a large number of samples. 

Validation is the process of proving that a method is acceptable for its intended purpose. It is important to note that it is the method not the results that is validated. The most important aspect of any analytical method is the quality of the data it ultimately produces. The development and validation of a new analytical method may therefore be an iterative process. Results of validation studies may indicate that a change in the procedure is necessary, which may then require revalidation. Before a method is routinely used, it must be validated. There are a number of criteria for validating an analytical method, as different performance characteristics will require different validation criteria. Therefore, it is necessary to understand what the general definitions and schemes mean in the case of the validation of CE methods (Table 1). Validation in CE has been reviewed in references 1 and 2. The validation of calibrations for analytical separation techniques in general has been outlined in reference 3. The approach to the validation of CE method is similar to that employed for HPLC methods. Individual differences will be discussed under each validation characteristic. 

Saz and Marina [148] published a comprehensive review on HPLC methods and their developments to characterize soybean proteins and to analyze soybean proteins in meals. In the case of soybean derived products, a number of papers dealing with cultivar identification [149,150], quantification of soybean proteins [151-154], detection of adulteration with bovine milk proteins [151,155-158], and characterization of commercial soybean products on the basis of their chromatographic protein profile [159,160] have been published in the last years. Other studies deal with the analysis of soybean proteins added to meat [161-165], dairy [151,165-167], and bakery products [156,163,168,169]. The same research group developed perfusion RP-HPLC methods for very rapid separation of maize proteins (3.4 min) and characterization of commercial maize products using multivariate analysis [170], and for the characterization of European and North American inbred and hybrid maize lines [171]. 

In conclusion, for low-dose dmg products, it is important to be aware of the possibility that adsorption of the dmg from the sample solutions onto surfaces can lead to low or variable assay results. These surfaces include filters, volumetric flasks, and sample vials. Evaluation of the components that come in direct contact with the sample solutions for potential dmg adsorption should be conducted as part of method development. This is especially true for compounds containing active sites such as amino groups, as described in this case study. While, most potency analyses involve the use of some organic solvents in the dissolving solvent, dissolution analyses could be problematic since the compendial media are aqueous buffers. For components such as HPLC vials, it is important to examine not only the type... 

CASE STUDY 3 HPLC PURITY METHOD DEVELOPMENT CHALLENGES FOR A FIXED COMBINATION PRODUCT CONTAINING A LOW-DOSE ACTIVE INGREDIENT AND A HIGH-DOSE ACTIVE INGREDIENT... 

CASE STUDY 3 HPLC PURITY METHOD DEVELOPMENT CHALLENGES 251... 

Method Development Time - A new method based upon GC or HPLC can often be developed in less than one month - particularly for new chemicals belonging to a previously studied class or structural type. IA method developments, on the other hand, may require several months or several years as one synthesizes hapten derivatives, conjugates to protein, immunise. an animal to obtain antibodies, optimizes key assay parameters, and validates the final procedures. This becomes much less of a disadvantage if a stock of antibodies is available for the hapten of interest, in which case method development time for IA can be measured in weeks or a few months. 

Case Study HPLC Methods in Early Development 152... 

This chapter reviews the use of HPLC in pharmaceutical analysis from drug discovery to quality control. The focus is on HPLC analysis of drug substances (DS) and products (DP) such as assay for potency, purity evaluation, and dissolution testing. A case study of the various HPLC methods used during early clinical development illustrates the versatility of this technique. Detailed descriptions of HPLC applications in pharmaceutical development and LC/MS analysis in drug discovery and bioanalytical studies can be found elsewhere.1-6 The regulatory aspects in pharmaceutical testing are covered in Chapter 9. 

CASE STUDY HPLC METHODS IN EARLY DEVELOPMENT... 

Direct HPLC enantioseparation techniques, which are free of many disadvantages of GC, indirect and chiral mobile phase HPLC methods, have gained unequivocal prevalence in bio-analytical studies. Several methods have been advanced so much that they allow enantiose-lective determination not only of the parent chiral drugs but also of their pharmacologically relevant metabolites [121]. As already mentioned above, a direct injection of biofluids offers several advantages in terms of analysis time and sample recovery. Precolumns packed with achiral or chiral packings, or with the recently developed so-called restricted-access packing materials, may be useful in this case. 

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