UHPLC/UPLC

Alternatively, ultraperformance—ultrahigh-performance LC (UPLC— UHPLC) systems have the benefit over conventional LC of increased peak resolution, which may be sufficient to separate some of the analytes of interest from matrix components. The validated method of Badawi et al. [90] is an example of the application of a matched UHPLC—UPLC— tandem MS system to the analysis of oral fluid for DUID purposes. The additional benefits of higher peak sensitivity and the ability to shorten the run time of the analysis offered by UPLC systems make them ideal for DUID testing. To take full advantage of UHPLC—UPLC, fast tandem—MS instruments are required to obtain sufficient data points across such narrow peaks. 

Figure 9.3 shows an impurity separation under conventional pressures with a 5 /mi particle, 2.1 x 150 mm column, and the same separation performed via UPLC using a 2.1 x 50 mm column with 1.7 /im particles. The run time was improved by a factor of six, with overall resolution comparable to that of the original separation on the 5 /an column. The application of UHPLC technology to impurity profile analysis can exert a significant impact on laboratory productivity by achieving a... 

Another potential benefit of UHPLC is its capability of solving the most challenging separation tasks in pharmaceutical analysis. Figure 9.4 shows a UPLC method developed to analyze pharmaceutical formulations used to treat the common cold. Cold products often contain multiple active ingredients to treat different symptoms and can contain decongestants, antihistamines, pain relievers, cough suppressants, expectorants, and numerous excipients of various polarities. The analysis of a total of 20 components was achieved within 10 min. 

To minimize unacceptable interruptions in highly regulated work flows, the smooth transfer of legacy methods from conventional to fast LC methods (via geometric transfer or method redevelopment) is a critical issue for implementing fast LC for pharmaceutical applications. Method transfer from HPLC to UPLC is discussed in detail in the literature.52,53 Moreover, method transfer software that provides parameter conversion between UHPLC and conventional HPLC is available from instrument vendors. 

Figure 23.5 Separation with UHPLC [after T. Moritz, Nestle Research Center, Lausanne, with permission see also S.J. Bruce et a .. Anal. Biochem., 372, 237 (2008)]. Conditions sample, extract from human plasma column, 10 cm 2.1mm i.d. stationary phase, C8 UPLC 1.7pm mobile phase, 0.5mlmin water-acetonitrile with 0.1% formic acid, linear gradient pressure, 600 bar detector, TOF-MS with positive ESI. The first peaks are amino acids and low-mass metabolites, the ones between 8 and 11 min are phospholipids (besides background peaks). 

FIGURE 2.2 UHPLC-MS of pesticide residues spiked in tea (0.1 mg/kg) obtained with an Aquity UPLC BEH Shield C18 (2.1 x 150 mm, 1.7 pm) (Waters) and a QqQ mass analyser (Quattro Micro API) working in MRM mode. The compounds separated are (1) Propamocarb (2) Primicarb (3) Carbofuran-3-hydroxy (4) Mevinphos (5) Acetamiprid (6) Thiofanox-sulphon (7) Spiroxamine (8) Triasulfuron (9) Bromoxynil (10) Promecarb (11) Triadimefon (12) Fenhexamid (13) Fenoxycarb (14) Clethodim 15 Flufenoxuron. (Adapted from Chen, G., Cao, P, and Liu, R. 2011. Food Chem. 125 1406-1411. With permission.)... 

VOO TAG profile, and checked the possibility of using this technique as a tool for VOO adulteration control. In this study, the sample preparation consisted, as was the case for the HPLC-MS methods previously described, of a simple dilution of the oils in 2-propanol. The separation was carried out by means of a UHPLC system with a packed column of the following characteristics 2.1 x 150 mm UPLC 1.7 pm BEH C18. 

FIGURE 16.3 Number of papers published each year in the field of flavonoids by UHPLC and UHPLC-MS, since 2006. Black bars were obtained with keywords UPLC or UHPLC and flavonoids, while white bars were obtained with key words UPLC-MS or UHPLC-... 

The book begins with an overview of the history, principles, and advancement of chromatography. It discusses the use of UHPLC techniques in food metabolomics, approaches for analysis of foodborne carcinogens, and details of UPLC-MS techniques used for the separation and determination of capsaicinoids. Chapters describe the analysis of contaminants in food, including pesticides, aflatoxin, per-fluorochemicals, and acrylamide, as well as potentially carcinogenic heterocyclic amines in cooked foods. 

Ultra high performance liquid chromatography (UHPLC) or ultra performance liquid chromatography (UPLC) is a quite novel technique, but it is one of the most popular nowadays, due to the fact that it is much more rapid than conventional LC. UHPLC systems can work at very high pressures and are compatible with stationary phases with particle sizes of around two micrometers or even smaller. Thus, the development of UHPLC methods [69,77, 80] or the conversion of HPLC to UHPLC methods is a current trend, due mainly to the speed of analysis and low solvent use. 

Most conventional pumps operate at pressures up to 400 bar, while some ultra-high pressure systems may operate at as high as 1000-1200bar. Ultrahigh-pressure LC (UPLC or UHPLC) requires special made columns and connections for the high pressures. The high pressures are a result of columns packed with very small particles. 

Quite a few of the recent, abovementioned STA or GUS techniques actually employed UPLC or UHPLC (29, 30, 36, 37) upfront mass spectrometry. However, the enhancement in chromatographic resolution produces very narrow (commonly 1-3 s wide) chromatographic peaks (38), which is only compatible with mass spectrometry cycle times at least threefold shorter (provided no polarity switching or alternated collision energies are used). 

Most frequently, high-pressure liquid chromatography (HPLC) is used in LC-MS work, while more recently ultrahigh-pressure liquid chromatography (UHPLC or ultra performance liquid chromatography, UPLC, as it is termed by one manufacturer) has come into use. For very low saiiple amounts, nanoLC, i.e., capillary LC, can directly be interfaced to nanoESI. 

There are several software and automated systems for HPLC method development and optimization, such as Drylab , Chromsword , and ACD/AutoChrom MDS, and others (43 7). Their principles can be applied to UHPLC. In addition. Waters Corp. (Milford, MA) has recently promoted Fusion Method Development software. Fusion Method Development software from S-Matrix integrates seamlessly with Water s ACQUITY UPLC and Empower 2 Chromatography software to automate method development. The software automatically generates instrument methods and sample sets. Another feature of this software is to visualize data by statistically fitting the results. However, it cannot generate simulated chromatograms at predicted conditions, like Drylab can. 

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