UPLC reproducibility

Results of, uPLC Evaluations of Retention Time and Peak Area Reproducibility... 

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 18-6. UPLC chromatography of benzodiazepines on a 14.5-cm x 50- xm column packed with l- xm polybutadiene-encapsulated non-porous zirconia particles. Eluent pH 7 buffer-acetonitrile 68 22 at 100°C. Peaks 1, uracil 2, clorazepate 3, fluni-trozepam 4, clonazepam 5, chlordiazepoxide 6, oxazepam 7, clorazepate 8, diazepam. (Reproduced from reference 53, with permission from Elsevier.)...

We monitored the reproducibility for a 10 pL injection of loperamide and ketocon-azole over a 12.5 h period in both rat plasma and homogenized rat brain (diluted 1 4 with water). The results are displayed in Figure 8.7 and demonstrate the overall improved reproducibility that has been observed for UPLC. This is important for determining potential matrix effects. Improved carryover has also been observed in most cases (<0.1%) precluding the need for multiple solvent blank injections [41],... 

Figure 6.11 UPLC QqTOF MS chromatograms of a tylosrn (4.1 p.g/kg, RSD = 1.5%, n = 3) incurred honey sample (CE—collision energy). Plots A and C traces of tylosin A. Plots B and D traces of tylosin B. Plots A2—spectrum at 4.42 min from plot A1 Plot B2—spectrum at 3.88 min from Bl plot C2—spectrum at 4.43 min from plot Cl. Plot D2—spectrum at 3.87 min from plot D1. Proposed fragmentation is based on the nitrogen rule and accurate mass measurement. (Reproduced and modified from Wang and Leung ° with permission from the Canadian Food Inspection Agency published by John Wiley Sons copyright 2007. Crown in the right of Canada.)...

A pair of cross-validated Waters Acuity UPLC instruments (Waters Corp., Milford MA) are used at Vitalea Science for metabolic analyses and have very high reproducibility. Figure 16.3 shows AMS quantitation for a single metabolite peak from urine separated on different days. AMS provides an uncertainty estimate for each measurement based on the number of recorded " C atoms (CV = 100/v ), and multiple measures (>3) are made for each fraction providing a normal standard deviation that often equals the counting statistics. There should be two of the six data replicates that do not overlap at the Ict uncertainties but there is only one, and that one occurs at the sharply rising start of the peak. Thus, these UPLCs appear reproducible with AMS quantitation to better than statistical accuracy. The LLOQ of chromatographic fractions is discussed in detail below. 

FIGURE 16.3 A Test of UPLC-AMS quantitative reproducibility shows a metabolite peak in urine samples separated by UPLC and measured by AMS on different days. Error bars represent the AMS counting uncertainty at la and should overlap only at 4 of the 6 points. The only nonoverlap occurs at the rising edge of the peak. 

Stage that requires high efficiency as well as speed, due to the complexity of the sample matrix, and hence it is particularly challenging to achieve the goals. Therefore, the development of a rapid, sensitive, and reproducible method has been required for separation and determination of capsaicinoid compounds. The addition of ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) method fulfilled these aforementioned demands and showed some complementary advantages to the conventional HPLC-MS, u-HPLC methods in terms of shorter analysis times, low sample volume, and much improved sensitivity [71]. Therefore, nowadays this UPLC-MS technique is routinely performed in pharmaceutical industries and related contract research institutes, laboratories concerned with biochemistry, biotechnology, environmental analysis, natural product research, and several other research fields. The UPLC-MS method has successfully been applied for the determination of n-DHC, C, DHC, h-C, and h-DHC present in the varieties of hot peppers [71]. 

The UPLC allowed a better resolution, an equal or better sensitivity according to gradient, and a better reproducibility of peak areas and retention times, but did not reduce the time required for analysis. Figures 10.8 and 10.9 show chromatograms of pure standards of vitamins A and E, respectively, obtained with HPLC and UPLC. 

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... 

Figure 3.11 Extracted ion chromatograms for miz 548.12 (glucuronides of midazolam) from the analyses shown in Figure 3.10, for (a) HPLC and (b) UPLC. Note the two metabolites, arising from glucuronidation at different hydroxylation sites in midazolam, that are well-resolved in (b) but not in (a). Reproduced from Plumb et al. Rapid Commun. Mass Spectrom. (2004) 18, 2331, with permission of John Wiley Sons, Ltd.

Figure 3.12 Sensitivity comparison of UPLC vs HPLC. Same sample (lOng/mL in rat plasma) was injected twice, once for HPLC/MS/MS (3.5 pm particles) and once for UPLC/MS/MS (1.7 pm particles) both columns were 2.1 x 50mm. Examples shown are for diphenhydramine (ESI+, left) and ibuprofen (ESI, right). Reproduced from Yu Rapid Commun. Mass Spectrom. (2006) 20,...

---