Pharmaceutical industry technique hyphenation

With the advent of new techniques, especially hyphenated techniques, through the combination of two well-known methods, better results and considerable timesaving can be achieved in order to ensure effective treatments capable of meeting the needs of pharmaceutical industry and of the patients. 

Hyphenated analytical techniques such as LC-MS, which combines liquid chromatography and mass spectrometry, are well-developed laboratory tools that are widely used in the pharmaceutical industry. Eor some compounds, mass spectrometry alone is insufficient for complete structural elucidation of unknown compounds nuclear magnetic resonance spectroscopy (NMR) can help elucidate the structure of these compounds (see Chapter 20). Traditionally, NMR experiments are performed on more or less pure samples, in which the signals of a single component dominate. Therefore, the structural analysis of individual components of complex mixtures is normally time-consuming and less cost-effective. The... 

The concurrent identification and quantification of organic impurities is a principal use of liquid chromatography in the pharmaceutical industry. However, the application of liquid chromatography to this task highlights a weakness of this technique when compared to gas chromatography specifically, the lack of a universal detector. Great strides have been made to create detectors and hyphenated techniques to address these problems. However, multiple detectors and analytical procedures may be necessary to accurately and specifically identify and quantify the impurities in complex systems. 

Natural products have been, and remain, a rich source of leads for the pharmaceutical industry and many marketed drugs are either natural products or are modifications of such substances. Hence, considerable effort is spent in isolating and characterising chemicals from natural sources which can be tested in a variety of biological screens. Often, it is necessary to carry out laborious extraction and purification steps and the advent of directly coupled HPLC-NMR has been explored as an alternative technique for natural product identification. The use of HPLC-NMR, and other hyphenated techniques such as HPLC-MS-MS, for identification of natural products from plant sources has been reviewed by Wolfender and co-workers [40,41],... 

During the last few years, more progress has been achieved by hyphenating LC-NMR to MS. The LC-NMR-MS or LC-NMR/MS (referred to as LC-MS-NMR in this chapter) has expanded the structure-solving capabilities by obtaining simultaneously MS and NMR data from the same chromatographic peak. There are some compromises that have to be taken into account because of the differences between MS and NMR, such as sensitivity, solvent compatibility, and destructive versus nondestructive technique, discussed below. LC-MS has been used for many years as a preferred analytical technique however, with the development of electrospray ionization techniques, LC-MS has been routinely used for the analysis of complex mixtures in the pharmaceutical industry. LC-MS-NMR is a combination of LC-MS with electrospray and LC-NMR presented below. 

The capability of analyzing a complex mixture in a chromatographic run by the hyphenation of several techniques, such as NMR and MS, to HPLC is becoming more popular in the pharmaceutical industry. NMR and MS data on the same analyte are crucial for structural elucidation. When different isolates such as metabolites are analyzed by NMR and MS, one cannot always be certain that the NMR and the MS data apply to the same analyte, especially when the analytes have been isolated using analytical columns and prep columns for the MS and NMR analysis, respectively. HPLC conditions are not always reproducible when analytical and prep-HPLC columns are used to isolate different amounts of the analytes of interest. To avoid this ambiguity, LC-MS and LC-NMR are combined. MS data should be obtained initially because with NMR, data collection in the stop-flow mode can take hours or days, depending on the complexity of the structure and the amount of sample. This is why it is preferable to designate this operation as LC-MS-NMR rather than LC-NMR-MS or LC-NMR/MS. 

Even though, there is no cookbook for HPLC method development this book provides several strategies that the reader could use when presented with a particular situation. These strategies could be stored as tools in the scientists method development arsenal, and drawn from when needed to tackle a particular separation. Moreover, some novel approaches for implementing HPLC, fast HPLC, and hyphenated HPLC techniques towards pharmaceutical analysis are discussed. This book has the potential to serve as a useful resource for the chromatographic community. It can be used as a handbook for the novice as well as the more experienced pharmaceutical chemist who utilizes HPLC as an analytical tool to solve challenging problems regularly in the pharmaceutical industry. 

Capillary isoelectric focusing (CIEF) is a high-resolution technique for protein and peptide separation performed at academic sites and in the biotechnology and pharmaceutical industries for the analysis and characterization of, for example, recombinant antibodies and other recombinant proteins, isoforms of glycoproteins, point mutations in hemoglobin, and peptide mapping. Also, hyphenation to mass spectrometry and chip-based CIEF (microfabrication) have shown promise. CIEF kits and specific recipes/application notes are available from vendors of capillary electrophoresis (CE) equipment, as are a vast amount of publications and handbooks of CE published over recent years. 

Recent reviews for hyphenated techniques have also been published,14 15 and the field continues to grow rapidly. As of writing the present chapter, Chemical Abstract Services lists over 2600 entries for LC-NMR alone The possibility of presenting an authoritative work on the whole topic of hyphenated characterization techniques in a single chapter would be, therefore, impossible. Instead, a few techniques have been chosen, based on the author s experience, to be common and useful. The vast majority of these examples are organic qualitative analyses at trace levels to address the commonly asked question in the pharmaceutical industry What is it . 

The first exposure to spectroscopy for most scientists is ultraviolet/ visible absorbance. As virtually every HPLC chromatograph employed in the pharmaceutical industry uses UV absorbance as the detection method, it is no wonder that the most popular hyphenated technique is HPLC-DAD. DAD spectrographs have been coupled to all liquid-based chromatographic systems including HPLC (preparative, analytical, and microbore), capillary electrophoresis (CE) and supercritical fluid chromatography (SFC). There have been several successes with TLC plates,18 but it is more common for developed plates to be scraped and the sample analyzed offline. 

ICP is seeing more use as a sample introduction system for various hyphenated techniques. New to the pharmaceutical industry is the use of inductively coupled plasma-mass spectrometry (ICP-MS). ICP-MS offers excellent versatihty and sensitivity to the analyst, and greatly complements any pharmaceutical... 

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