The Application Notebook-03-01-2017

This application note outlines a simple, fast, and cost‑effective QuEChERS-based method for the determination of limonin in citrus juice. Limonin is extracted from a variety of juice samples using acetonitrile and citrate-buffered salts. The sample extract undergoes cleanup by dispersive-SPE (dSPE) using primary‑secondary amine (PSA), C18, and graphitized carbon black (GCB) to remove unwanted matrix components, including sugars, acids, and pigments, and to yield a clear sample extract. Analysis is performed by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) using a Selectra® C18 HPLC column (although high performance liquid chromatography [HPLC]–UV can also be used).

Quality and consistency in reagents is critical to successful drug discovery and development. When targeting a particular protein of interest, in vitro experiments should be performed with proteins of biological properties similar to those for in vivo tests. It is important that molecularity, purity, shape, and degree of heterogeneity remain the same when any alterations are made to the model protein or the formulation buffer. Multi-angle light scattering (MALS) combined with size-exclusion chromatography (SEC-MALS) is a very useful technique to monitor the solution properties of the protein as changes to reagents are made.

In this application note, an antibody–drug conjugate (ADC) was analyzed using a TSKgel® Butyl-NPR column, the least hydrophobic of the TSKgel HIC columns. Both unconjugated and drug conjugated Trastuzumab samples were successfully separated with baseline resolution. The baseline resolution enabled an easy integration and quantification of different drug pay loads in ADC characterization.

Projects in drug discovery and safety constantly aim at development of novel and safer drugs, therapeutics, and diagnostics. During active pharmaceutical ingredient (API) development, drug stereoisomerism is recognized as an issue having clinical and regulatory implications. Enantiomers have essentially identical physical and chemical properties, while potentially showing large differences in toxicity.

Wyatt TechnologyReversed-phase chromatography represents one of the most popular applications of high performance liquid chromatography (HPLC), with particular importance for protein characterization. Because elution depends on the hydrophobicity of the sample, it is generally impossible to identify the separated products on the basis of their elution time (volume). Frequently, each eluted fraction must be isolated further and analyzed with other techniques to gain some understanding of the molecular behaviour of the protein.