Examples, reversed HPLC method development

The six chromatographic modes available in HPLC for the isolation and purification of natural products are described in more detail in Section 9.02.2. Since the groups of natural products differ in their molecular properties, certain chromatographic modes have been shown to work better with particular natural product groups. However, in order to take full advantage of a specific HPLC mode for a separation task and to effectively utilize time and resources, comprehensive method development should be performed. An example of such method development from the analytical to the preparative stage is described for reversed-phase chromatography (HP-RPC), the most frequently employed mode in natural product purification, in Section 9.02.3. 

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... 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

Successful separations can be carried out only by planning and careful experimentation, the details of which are discussed extensively in Chapter 5. In one sense there are too many ways to achieve a separation in LC. But while this makes the first choice of where to start difficult, the good news is that there are many ways to achieve success. By looking at Figure 4-1 it is obvious that the use of the reverse-phase mode in LC has broad applicability and is, in fact, the most used mode of LC. Reverse phase is used for 80-85% of the separation problems encountered by users of HPLC. For this reason, the majority of Chapter 5, on developing methods, is devoted to reverse-phase examples. Additionally, Chapter 11 is a useful experiment to experience the method development aspect of this mode. Chapter 9 is a useful experiment to experience method development in the normal phase mode. 

Analysis of Target Compounds. Matching El or Cl spectra and LC retention times to those obtained via analytical standards is exactly analogous to GC/MS methods development. Thus the major effort involves the determination of an appropriate HPLC column for any given analyte or analyte class. For example, conventional reversed phase HPLC columns are useless for extremely polar compounds such as sulfonic and certain carboxylic acids ion exchange based columns are more appropriate. 

Successful enantioseparation of individual N -protected amino acids stimulated the development of a rapid method of their simultaneous enantioseparation and quantification in a mixture. A feasibility study on this topic has been recently published by Welsch et al. [69]. The two-dimensional HPLC method involves online coupling of a narrow-bore C18 reverse phase (RP) column in the first dimension (separation of racemic amino acids) to a short enantioselective column based on nonporous 1.5 pm particles modified with t-BuCQD in the second dimension (determination of enantiomer composition). Using narrow-bore column resulted in fast analysis time for example, the mixture of nine racemic N-DNB-protected amino acids was completely analyzed within 16 min. 

HPLC techniques have also been used in the determination of log P values. Lambert et al. (1990), for example, have described the development of a preformulation lipophilicity screen utilizing a C18 derivatized HPLC column. They appeared to prefer this column to the traditional reverse-phase HPLC columns, which may yield a poor correlation between log P and the capacity factor (k ). A potential problem with the use of HPLC retention data is that it is not a direct method and thus requires calibration. Futhermore, there may be problems with performing experiments above pH 8. 

An example of a simple CZE method for peptide analysis and characterization is the one developed for protegrin IB-367.37 IB-367 is a peptide containing 17 amino acid residues that possess antimicrobial properties, and it is being developed for treatment of oral mucositis associated with aggressive cancer chemotherapy as well as other topical applications. This polycationic product was chemically synthesized using solid-phase and purified by preparative reversed-phase HPLC. IB-367 is rich in cysteine and arginine residues. 

LC-NMR can be used to identify natural products in crnde plant extracts that usually consist of complex mixtnres. The crnde natural product extracts normally contain a great nnmber of closely related and difficult-to-separate compounds. The classical separation approach may become very tedious and time-consuming. The directly conpled HPLC-NMR presents an efficient separation techniqne together with a powerfnl spectroscopic method to speed up the identification process. LC-NMR has been nsed extensively for characterization of natnral prodncts. More recently, the combination of LC-NMR and LC-MS has been further developed in this field. Eor example, Wilson et al. have nsed combined on-flow NMR and electrospray ionization MS to characterize ecdysteroids in extracts of silene otites. After reversed-phase HPLC nsing D2O in acetonitrile-dj and UV detection, the LC flow was split 95 5 for the simnl-taneous detection by NMR and MS. The peaks of interest were analyzed by stop-flow NMR to give better quality spectra for structural assignment. 

HPLC has been one ofthe most widely used analytical methods for determining PAHs in complex environmental samples. The development of a chemically nonpolar stationary phase for HPLC has provided a unique selectivity for separation of PAH isomers that are often difficult to separate by GC columns. For example, chrysene, benz[a]anthracene, and triphenylene are baseline resolved with a C-18 reverse phase column packing. A detection limit of subpicogram to picogram levels of PAHs per sample has been achieved by HPLC with fluorescence detector (For and Staley 1976 Furuta and Otsuki 1983 Futoma et al. 1981 Golden and Sawicki 1978 Lawrence and Weber 1984 Marcomini et al. 1987 Miguel and De Andrade 1989 Nielsen 1979 Risner 1988 Tomkins et al. 

Reversed-phase HPLC is the method of choice for the quantitative analysis of procyanidins. In view of the known instability of procyanidins and the problem of developing suitable sample clean up procedures direct analysis of crude extracts is probably the best approach for quantitation. However, the separation capacity of HPLC in combination with the most commonly used UV detection at 280 nm is generally insufficient to generate useful quantitative results. Direct chromatographic determination of procyanidins in qualitative analysis has been frequently performed for example in the analysis of wine [168,252], beer [32], grape seeds [28], rhizomes of tormentil (Potentilla tormentilla) [253], Sesbania sesban... 

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