App Notes

Hops, Humulus lupulus, are one of the primary ingredients in beer and serve as both a natural preservative and as a flavoring agent. These leafy green flowers, shown in Figure 1, are responsible for the characteristic bitterness in beer, but can also impart other flavors such as floral, tangy, piney, or citrusy notes. One factor that impacts the eventual flavor profile of beer is the selection of hop variety, as different strains lead to different flavors and aromas. Another important aspect is the timing of the hop addition during beer brewing. Brewing is a multi-step process that begins by mixing grains with hot water to convert starches in the grain to a sugary solution called wort. The wort is filtered and then boiled together with hops and other specialty ingredients to further develop flavors. At the completion of the boil, yeast is added to initiate fermentation. Hops can be added at any point during or after the boil to bring out desired flavors. Generally, hops are added earlier to draw out bitterness and later to highlight aroma and flavor.

Metabolomics and its toolset provide a foundation for quantitative biology and are indispensible for the detection of small molecules produced and/or transformed in the cells of living organisms.1,2 Different estimates indicate that a majority of differentially expressed analytes remain unknowns. The high sensitivity, peak capacity and reproducibility of GC-MS have made it one of the most widely used techniques for plant and animal metabolite profiling. Time-of-flight mass spectrometry (TOFMS) provides additional benefits such as reduced analysis times, effective peak deconvolution and an ability to interrogate rich data sets repeatedly for novel materials.

Liver disease affects more than 800 million people worldwide causing at least 1.5 million deaths annually. Alcohol consumption and alcoholic liver disease (ALD) continue to be a major cause of morbidity and mortality in the US. Polychlorinated biphenyls (PCBs) are persistent environmental pollutants. Exposures to PCBs have been associated with non-alcoholic fatty liver disease. Monitoring these and other diseases of the liver can be achieved by evaluating metabolites in serum or plasma but a key, basal understanding can come from evaluation of the diseased tissues themselves. Changes detected here will help understand the biology and physiology and may translate to the circulation. Mice provide a good model for this type of research.

The continuous influx of new synthetic drugs such as cannabis analogs into society is a major problem for law enforcement, forensic laboratories, and the medical community.1,2 Relatively simple organic transformations produce novel and licit psychotics that can elude detection by standard analytical methods.3,4 Detection and characterization of synthetic drugs is complicated by 1) the wide range of active ingredients and variety of botanical matrices, 2) the rate at which new drugs and blends appear on the market, 3) the fact that these synthetic drugs and metabolites are often not targeted during routine forensic analyses,5,6 and 4) these newly emerging compounds are typically not present in commercially available mass spectral libraries. High performance time-of-flight mass spectrometry is a practical choice for the analysis of these moving targets.

UHPLC columns can significantly improve chromatographic separations, but they also present unique challenges. Once the UHPLC system components are optimized, perhaps your greatest concern is protecting the column from the damaging effects of microparticulates and sample contaminants. An ultra-high performance column protection system, specifically designed for UHPLC systems using sub-2 μm and core-shell particle columns, can be used to extend column lifetime (saving both money and time through less frequent column replacement), while minimizing system troubleshooting and downtime.

Nicotinic acid and nicotinamide were extracted from human plasma by performing a rapid protein precipitation using Impact Protein Precipitation Plates followed by HPLC analysis using a Gemini 3 μm C18 100 x 4.6 mm HPLC column and positive polarity ESI LC/MS/MS system. Impact technology offers easy, fast protein removal while providing maximized recovery of the target analytes. The Gemini 3 μm C18 HPLC column produced excellent chromatographic resolution, sensitivity, and high peak capacities.

A new high efficiency GFC column, Yarra, was recently introduced and is significantly more efficient than other GFC columns on the market. In addition to higher efficiency, Yarra columns demonstrate significantly higher inertness to ionic interactions versus other GFC columns; however, such chemical characteristics sometimes require changes to operating parameters. Performing method development for protein aggregation analysis using next-generation Yarra GFC columns will be discussed.

Protein precipitation is compared to a phospholipid removal product, Phree Phospholipid Removal Plates, to assess the cleanup capabilities of each technique. By measuring for total phospholipids using the 184  184 mass transition, LC/MS/MS analysis indicates that Phree Phospholipid Removal Plates result in significantly cleaner samples, reduced ion suppression, and extended HPLC/UHPLC column lifetime. The technique followed a simple procedure that is similar to a traditional protein precipitation, required no method development, and can be automated to provide higher throughput.

Gel Permeation Chromatography (GPC) is principally used to separate polymers and organic molecules based on their size. An important aspect in optimizing GPC separations is column selection, especially when multiple columns are used. Mobile phase selection and column temperature are also important factors that must be considered when developing a method, as they can influence the solubility and viscosity of diluent and analyte respectively. Column, mobile phase, and temperature selection will be discussed to optimize GPC methods using Phenogel GPC/SEC columns.

Kinetex 5 μm core-shell technology columns provide chromatographers a simple solution for dramatically improving the Performance of their methods developed on 5 μm fully porous columns. This newly introduced core-shell media delivers backpressures of a fully porous 5 μm particle at efficiencies equal to or better than a fully porous 3 μm particle. Without the need for extensive method development, replacing the fully porous 5 μm column with the Kinetex 5 μm core-shell column results in improved chromatographic resolution and sensitivity. In addition, the lower backpressure can provide many benefits such as longer column lifetime, higher throughput, and increased system compatibility.