UHPLC conditions

In UHPLC conditions, solvents become compressible. Consequently, a significant portion of the piston stroke is used for compressing the solvent. Owing to this fact, it is advantageous to run the primary HPLC pump at high flow rates until the desired secondary operating pressure is achieved. As well, both compression and decompression... 

The total analysis time for the determination of the studied compounds under these UHPLC conditions was between 12.5 min [18,28,29] and 20 min [27] for the analysis of cocoa samples, and between 3.1 min [40] and 12.5 min [30-39] for the analysis of biological samples. Although the procyanidins and alkaloids and their metabolites were eluted in a shorter analysis time, some time was necessary to clean up the column with phase B and reequilibrate the colunm to return to the initial conditions. 

Core-shell silica or fused-core particle (depending on manufacturer) 2.7 pm (1.7-pm solid core, 0.5-pm porous shell) (1.7-pm particles also available) Achieves UHPLC conditions with an HPLC (2.7-pm particles) Smaller 1.7-pm type used for UHPLC ... 

In the first case study, acebutolol was analyzed in the presence of acetaminophen, nortriptyline, and qunidine (each spiked at a level of 0.1% relative to acebutolol). Results are listed in Table 2.1. Note that shallower (compared to the base HPLC method) gradient slopes (and thus longer than expected run times) were needed under UHPLC conditions to maintain resolution. 

The theoretical relationships that govern the scaling of methods from HPLC to UHPLC conditions are well established and provide a good framework for the adjustment of other chromatographic parameters as stationary phase particle size... 

Recently, several chromatography columns packed with SPP stationary phases have become commercially available. With the advent of these phases, chromatographic efficiencies associated with UHPLC conditions can now be achieved on many traditional HPLC platforms. Because of this capability, translating UHPLC methods to traditional HPLC platforms can better make use of the SPP technology. 

The traditional solution to poor resolution has been to increase the run time, which negatively affects the analytical throughput without completely solving the issues. An alternative solution consists of using columns packed with sub-2 p.m particles under UHPLC conditions. Reducing the particle size leads to significant improvements... 

Isocratic separation of test compounds is a useful way to demonstrate the performance of a system. Basic chromatographic characteristics, such as theoretical plates, are easily measured and can be compared to what is expected from theory and to performance of other chromatographic systems. Figure 17-4 is a UHPLC chromatogram obtained under isocratic conditions on a 43-cm-long capillary column packed with 1.0-pm nonporous Cl 8 particles (Eichrom... 

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). 

The hyphenation of UHPLC to MS, carried out generally with electrospray (Table 2.1), requires volatile and low-viscosity solvents to favor the ionic evaporation, as was the case in HPLC-MS, and to keep the pressures low, which is particularly important in UHPLC. These requirements restrict the composition of the mobile phase however, the separation of high number of compounds is still achieved. The conditions compiled in Table 2.1 show that the aqueous phase is generally constituted by water, 0.02-0.2% formic in water, or 2-10 mM ammonium acetate, and methanol or acetonitrile are the solvents chosen for the organic phase, like in conventional LC. The separation has been carried out at a higher temperature than room temperature, up to 50°C [75], to keep viscosity of the solvent low, which reduces the pressure in the chromatograph. 

In the following sections, the sample pretreatment techniques and the UHPLC-MS/MS conditions for determining procyanidins and alkaloids in cocoa samples and their metabolites in biological samples are reported. 

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