Reversed-phase Ultra-performance Liquid Chromatography Research Articles

Evaluation of the repeatability of ultra-performance liquid chromatography–TOF-MS for global metabolic profiling of human urine samples

The application of reversed-phase ultra-performance liquid chromatography, based on the use of sub 2 μm particles, combined with time-of-flight mass spectrometry has been investigated for the production of global metabolite profiles from human urine. The stability and repeatability of the methodology, which employed gradient elution, was determined by the repeat analysis of a pooled quality control (QC) sample. As seen in previous studies conducted with conventional LC–MS an element of system conditioning was required to obtain reproducible data, as the initial injections were unrepresentative. However, once the system had equilibrated excellent repeatability in terms of retention time, signal intensity and mass accuracy was seen providing confidence that for this matrix, the within-day repeatability of UPLC–TOF-MS was sufficient to assure data quality in global metabolic profiling applications.

Quantitative Estimation of Triterpenoids and Formononetin in Rhizomes of Black Cohosh (A. racemosa) and Dietary Supplements that Claim to Contain A. racemosa by Reversed Phase Ultra-Performance Liquid Chromatography and Evaporative Light Scattering Detection

Actaea racemosa (Black cohosh) is one of the most extensively studied herbal products, and is used as an herbal dietary supplement for the alleviation of menopausal symptoms [1]. The rhizomes/roots used in black cohosh products are often collected from the wild; correct identification is therefore crucial. Actein, 23-epi-26-deoxyactein (syn. 27-deoxyactein), acetylshengmanol 3-O-xyloside, cimigenol 3-O-arabinoside, and cimigenol 3-O-xyloside represent the major triterpene glycosides found in black cohosh [1]. Black cohosh has been described by previous studies to contain a phytoestrogen, formononetin. A UPLC-ELSD method has been developed for the analysis of major triterpenoids and formononetin in Actaea racemosa samples. The best results were obtained with Acquity UPLC™ BEH C18 (100 mm × 2.1 mm, i.d., 1.7 µm) column system using an isocratic elution with a mobile phase consisting of water and acetonitrile:methanol (7:3) at a constant flow rate of 0.3 mL/min. Owing to their low UV absorption, the triterpene saponins were detected by evaporative light scattering. Within 5.5 minutes three main glycosides [cimiracemoside (2), 26-deoxyactein (3) and actein (4)] and the isoflavonoid formononentin (1), could be separated, with detection limits of 5, 5, 10 and 0.1 µg/mL, respectively. The method was successfully used to analyze different Actaea recemosa market products, as well as to distinguish between other A. species. For the products containing black cohosh and in rhizomes of black cohosh, there was a significant variability in the amounts of the selected triterpene glycosides and formononetin was not detected. Acknowledgements: This research is funded in part by “Science Based Authentication of Dietary Supplements” Funded by the Food and Drug Administration grant number 2 U01 FD 002071-07 and the United States Department of Agriculture, Agricultural Research Service Specific Cooperative Agreement Number 58-6408-06-067.

Determination of Fluorescence-Labeled Asparaginyl-Oligosaccharide in Glycoprotein by Reversed-Phase Ultraperformance Liquid Chromatography with Electrospray Ionization Time-of-Flight Mass Spectrometry

Eight fluorescence reagents, i.e., DBD-F, NBD-F, DNS-Cl, NDA, PSC, FITC, Fmoc-Cl, and DMEQ-COCl, which are reactive to an amino functional group, were tested for the labeling of asparaginyl-oligosaccharides in a glycoprotein. Although the optimal reaction conditions and the fluorescence maximal wavelengths were different for each reagent, the highly sensitive fluorescence detection at the femtomole level of Disialo-Asn (a representative asparaginyl-oligosaccharide) was obtained from the labeling utilizing these reagents. Among them, PSC was the most reliable reagent in terms of detection sensitivity (approximately 3 fmol, signal-to-noise ratio of 5 (S/N = 5) on the chromatogram). However, the structural information could not be obtained from the fluorescence detection. Thus, the on-line determination of a real sample was carried out by UPLC-ESI-TOF-MS. The detection limit of the PSC-labeled Disialo-Asn by selected-ion chromatography was 58 fmol (S/N = 5). When the proposed procedure was applied to the determination of oligosaccharides in ovalbumin, 15 species of PSC-labeled oligosaccharides possessing Man, GlcNAc, and Gal units were identified from the UPLC-ESI-TOF-MS. The number of identified oligosaccharides was relatively greater than the method using Fmoc-Cl. Based on the ovalbumin results, the proposed labeling with PSC followed by UPLC-ESI-TOF-MS detection seems to be useful for the on-line asparaginyl-oligosaccharide analysis.

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers: Theoretical and practical implications for the separation of oligonucleotides

Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 μm C 18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results.

Rapid Reversed Phase Ultra-Performance Liquid Chromatography Analysis of the Major Cocoa Polyphenols and Inter-relationships of Their Concentrations in Chocolate

Chocolate and other cocoa-containing products are a rich source of polyphenols. This paper describes an ultra-performance liquid chromatography (UPLC) method that can separate and quantify in 3 min six of the major chocolate polyphenols: catechin; epicatechin; B2 (epicatechin-4beta-8-epicatechin); B5 (epicatechin-4beta-6-epicatechin); C1 (epicatechin-4beta-8-epicatechin-4beta-8-epicatechin); and tetramer D (epicatechin-4beta-8-epicatechin-4beta-8-epicatechin-4beta-8-epicatechin). A survey of 68 chocolate samples indicated that there was a strongly predictive relationship between epicatechin and the other individual polyphenols, especially procyanidin B2 (R 2 = 0.989), even though the chocolates came from varied sources and manufacturers. The relationship was less strong with catechin, and so further work to explore the reasons for this difference was performed. Chiral analysis on a subset of 23 chocolates showed that (-)-epicatechin had a predictive relationship with (+)-catechin in line with the other polyphenols, but not with (-)-catechin (the predominant form). This indicates that (-)-catechin is the most affected by manufacturing conditions, possibly formed through epimerization from (-)-epicatechin during processing. The results show that epicatechin concentrations can be used to predict the content of other polyphenols, especially B2 and C1, and total polyphenols content. Finally, the (-)-catechin content is not predictable from the epicatechin content, and it is concluded that this is the main form of polyphenol that varies according to manufacturing conditions and cocoa origin.

A comprehensive urinary metabolomic approach for identifying kidney cancer

The diagnosis of cancer by examination of the urine has the potential to improve patient outcomes by means of earlier detection. Due to the fact that the urine contains metabolic signatures of many biochemical pathways, this biofluid is ideally suited for metabolomic analysis, especially involving diseases of the kidney and urinary system. In this pilot study, we test three independent analytical techniques for suitability for detection of renal cell carcinoma (RCC) in urine of affected patients. Hydrophilic interaction chromatography (HILIC–LC–MS), reversed-phase ultra performance liquid chromatography (RP–UPLC–MS), and gas chromatography time-of-flight mass spectrometry (GC–TOF–MS) all were used as complementary separation techniques. The combination of these techniques is best suited to cover a very large part of the urine metabolome by enabling the detection of both lipophilic and hydrophilic metabolites present therein. In this study, it is demonstrated that sample pretreatment with urease dramatically alters the metabolome composition apart from removal of urea. Two new freely available peak alignment methods, MZmine and XCMS, are used for peak detection and retention time alignment. The results are analyzed by a feature selection algorithm with subsequent univariate analysis of variance (ANOVA) and a multivariate partial least squares (PLS) approach. From more than 2000 mass spectral features detected in the urine, we identify several significant components that lead to discrimination between RCC patients and controls despite the relatively small sample size. A feature selection process condensed the significant features to less than 30 components in each of the data sets. In future work, these potential biomarkers will be further validated with a larger patient cohort. Such investigation will likely lead to clinically applicable assays for earlier diagnosis of RCC, as well as other malignancies, and thereby improved patient prognosis.