UPLC™ system

Some, uPLC systems are equipped with UV absorbance detection, and other systems allow for both UV absorbance and fluorescence detection. Fluorescence detection increases the sensitivity and selectivity of certain applications and is the method of choice in many separation-based assays. The liquid (mobile phase + sample) leaving the individual flow cells designated for UV detection is transferred through capillaries to a bank of 24 flow cells designated for fluorescence detection. 

FIGURE 6.12, uPLC system with time trigger fraction collection capabilities. 

Aqueous solubility values for the samples analyzed compared favorably with results obtained by traditional methods. The solubility values for amiodarone HC1, reserpine, and benzanthrone were lower than the LOQ of the, uPLC system used for the evaluation. Results of the evaluation of compound solubility employing no-filtration /iPLC were compared with those obtained by two traditional methods (1) multiscreen filtration followed by a UV plate reader, and (2) the shake flask method followed by a UV plate reader. As shown in Figure 6.31, the solubility values determined by the different methods are comparable for most compounds examined. Figure 6.32 shows the results of evaluations of aqueous solubility at four different pH levels for phenazopyridine and piroxicam samples. 

A Waters Acquity UPLC system with a cooling autosampler and column oven was used. The stationary phase was a Waters Acquity BEH C18 column (50 x 2.1 mm, 1.7 /.un particle size). The column was maintained at 40°C. The mobile phase consisted of water and acetonitrile, each containing 0.3% formic acid and was delivered at 0.35 mL/min in a gradient mode at 60% water from 0 to 1.5 min, linearly decreased to 10% water in 0.5 min, and then returned to 60% water. Sample vials were maintained at 4°C. 

Lercanidipine — Kalovidouris et al.57 applied UPLC-MS/MS to the determination of lercani-dipine in human plasma after oral administration of lercanidipine. A Waters Acquity UPLC system with cooling autosampler and column oven was coupled with a Waters BEH C18 column (50 x 2.1 mm, 1.7 jum). The mobile phase was composed of 70% acetonitrile in water containing 0.2% v/v formic acid, delivered at a flow of 0.30 mL/min. The column temperature was maintained at 40°C and sample vials at 5°C. 

FIGURE 5 (a) Peptide digest run on a 4.8-gm particle on a traditional HPLC system. Peak count is 70, and peak capacity is 143. (b) Peptide digest run on a 1.7-gm particle on the ACQUITY UPLC system. Peak count is 168, and peak capacity is 360. (Courtesy of Waters Corp.)... 

The detectors used with UPLC systems have to be able to handle very fast scanning methods because peak half-height widths of around 1 s are typically obtained with columns packed with 1.7-p.m particles. In order to accurately and reproducibly integrate an analyte peak, the detector sampling rate must be high enough to capture enough data points across the peak. Conceptually, the sensitivity increase for UPLC... 

Figure 1.2. (a) The traditional technique of low-pressure liquid chromatography using a glass column and gravity-fed solvent with manual fraction collection, (b) A modern automated HPLC instrument (Waters Acquity UPLC system) capable of very high efficiency and pressure up to 15,000 psi. 

Bolechova, Caslavsky, Pospichalova, and Kosubova report a protocol for the extraction of selected pyrrolizidine alkaloids (PAs) in feed. They determined PAs using an ultraperformance liquid chromatography (UPLC) system coupled to a tandem mass spectrometry equipped with an electrospray interface (ESI). Their steps to extract alkaloids for this determination are as follows ... 

A coupling between the UPLC system and a mass spectrometer enables chemists to combine the physical separation capabilities of liquid chromatography with... 

In both systems, the mobile phase was a binary gradient mixture of 30 mM potassium phosphate buffer at pH 2.3 and acetonitrile. In the UPLC system, a binary mixture of 10 mM formic acid and acetonitrile was also tested at the mobile phase. Folic acid was quantified using UV detection at 290 nm, whereas other forms were quantified by using fluorescence detection excitation 290 nm and emission 360 mn for H4folate, 5-CH3-H4folate, and 5-HCO-H4folate, and excitation 360 nm and emission 460 nm for lO-HCO-folic acid. 

The UPLC system did not offer shorter analysis times. This could be explained by the difference in the column s chemistry, as well as the proportion of water in the mobile phase. However, it gave the best reproducibility of retention time... 

Overall, LC-MS analysis with a reversed-phase column is very powerful and common for the analysis of individual species after fractionation of an interest class, whereas a UPLC system is popularly used for global lipid analysis. Regarding the MS detection, the former is more associated with an MRM approach while the SIM approach is the likely choice in the latter case. 

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