Development of Fast HPLC Methods

Reducing chromatographic media particle size allows the number of theoretical plates per second to be increased. However, due to the resolution 

HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright 2007 by John Wiley Sons, Inc. 

To understand how to optimize a separation for speed, it is worth revisiting some of the theoretical concepts developed earlier in this text. The analysis time, ta, is the time it takes for all sample components to elute off a column at a certain flow rate and is given by 

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Hertog et al. (119) developed a fast HPLC method for the identification and quantification of five major flavonoid aglycones (quercetin, kaempferol, myricetin, luteolin, and apigenin) in freeze-dried vegetable and fruits. However, due to the inadequate resolution of quercetin and luteolin on RP-HPLC on Nova-Pak C]8, two different eluents of different solvent strength and viscosity were utilized. The conditions for hydrolysis and extraction were tested based on different conditions of hydrochloric acid concentration (1.2-2.0 M), reaction period (0.5-6 h), and meth-... 

HPLC is a very powerful technique for qualitative and quantitative analysis. In the support of process development, HPLC plays an important role in monitoring a reaction, since each reaction component can be quantitated. In this role, the HPLC method must be fast, rugged, and specific, capable of separating all reactants, products, and byproducts. Development of appropriate analytical methods is often a rate-limiting step in process development. 

As we have seen so far, libraries of hydrogenation catalysts are never composed of more than a few dozen members, up to 100 to 200 at the most. Consequently, modern analytical equipment such as gas chromatography (GC) or high-performance liquid chromatography (HPLC) equipped with an auto-sampler or even flow-through NMR systems are sufficient to handle the analysis of the entire library. Nevertheless, a few groups have initiated research towards the development of fast, sometimes parallel, analytical procedures. A few reviews have appeared on this subject [59]. Here, we will concentrate on the methods developed to analyze hydrogenation reactions, or methods that could likely be applied. 

Ketamine, a general anesthetic producing hallucinations and exhibiting an addiction potential, and norketamine have been determined in urine using SPE combined with RPLC-ESI(+)-MS-MS [49]. Cheng and Mok [49] developed a fast HPLC-MS-MS method able to separate and detect ketamine in a 2.5 min chromatographic run with a LOD of 5ng/mL. The method was applied to routine ketamine urine screening to a maximum of 200 samples per day. 

Development of fast, accurate, and reproducible high-performance liquid chromatography (HPLC) methods has offset the use of traditional open-column and TLC methods in modern chlorophyll separation and analysis. A number of normal and reversed-phase methods have been developed for analysis of chlorophyll derivatives in food samples (unit F4.4), with octadecyl-bonded stationary phase (C]8) techniques predominating in the literature (Schwartz and Lorenzo, 1990). Inclusion of buffer salts such as ammonium acetate in the mobile phase is often useful, as this provides a proton equilibrium suitable for ionizable chlorophyllides and pheophorbides (Almela et al., 2000). 

The objective of blend analysis is to determine whether the blend is uniformly mixed or not (very similar to CU, except BU is for powders and CU is for dosage units). What one needs is quick answers during blend analysis, so fast and simple methods are preferable. There is no need to develop a new HPLC method for blend analysis, since the CU method can easily be employed here. 

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. 

Bisphenol A is used as a raw material to make polycarbonate and epoxy adhesives and can coatings. It is also used in flame-retardants, in unsaturated polyesters and in polyacrylate resins. Many foodstuff containers are made of these resins, including containers for oven and microwave cooking. Recent studies have shown that bisphenol type compounds have both mutagenic and cytotoxic properties [84]. Nerin et al. developed a fast screening method based on SPME and HPLC with fluorescence detection suitable for the analysis of several bisphenol derivatives and their degradation products in aqueous canned foods such as tuna, olives and corn [85]. The best results were obtained with carbowax and PDMS/DVB fibers. The detection limits were between 0.7 and 2.4ngmL while RSDs were between 14 and 32%. After the extraction parameters were optimized, the method was applied to... 

Mobile phase pH is one of the most powerful tools for modifying selectivity of compounds in HPLC. A systematic selection process for pH can make the difference between a fast development of a robust method and a long development process that may result in a separation that cannot be reproduced. While it is usually impractical to measure values of species prior to method development,... 

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