Examples, reversed HPLC method

Bohman and colleagues described a reverse-phase HPLC method for the quantitative analysis of vitamin A in food using the method of standard additions. In a typical example, a 10.067-g sample of cereal is placed in a 250-mL Erlenmeyer flask along with 1 g of sodium ascorbate,... 

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). 

In reversed-phase HPLC on a Cl8 column, only six peaks are usually found, and their order of elution is 5-tocotrienol, (3- and y-to-cotrienols, a-tocotrienol, 5-tocopherol, (3- and y-tocopherol, and a-tocopherol. Figure D 1.5.4 is an example of a reversed-phase chromatograph of tocopherols and tocotrienols in rice bran oil. The tocols with unsaturated side chain have shorter retention time than those with saturated side chain. The methyl substituents on the chromanol ring also affect the retention times of tocopherols and tocotrienols. However, the effect is reversed, compared with the normal-phase HPLC method. 

Another example of an HPLC method using clonazepam as the internal standard is available for the study of the stability of diltiazem tablets in the presence of its degradation products. The method utilizes a Micropak MCH-5 reversed-phase column... 

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. 

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. 

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... 

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