Application Notes: General

An effective UHPLC-MS method for high throughput separation, identification and quantitation of pseudoephedrine was developed on a Hypersil GOLD™ PFP 1.9 µm, 2.1 x 100 mm column.

The use of mobile phase pH to control analyte ionization states (pH-LCâ„¢) in reversed phase HPLC separations is a highly effective way to change selectivity. The ionized species of an analyte is shown to have higher polarity (less hydrophobicity) than the neutral species, which results in a loss of expected retention for that analyte. This can be attributed to less interaction with the hydrophobic stationary phase and greater affinity with the aqueous portion of the mobile phase. Ionized species also participate in ionic interactions with exposed and activated silanols, which impact peak shape and reproducibility.

Size exclusion chromatography is a powerful tool for the characterization of molecules differing in size and molar mass. It is widely used and well known for the determination of molar mass distributions and molar mass averages. For membranes GPC-SEC is a useful characterization method as it can measure the membrane characteristic pore size distribution, average pore size and molecular cut-off significantly faster than other methods.

In the last few decades, the novel functions of polysaccharides have provided a major impetus for increasing scientific attention. Among the most promising aspects are their immunomodulatory and antitumor effects, thickening agents and stabilizer effects.

Glucosamine (GlcN) is a major structural component in the biosynthesis of glycosaminoglycans, compounds involved in normal joint function. Use of GlcN as a dietary supplement in the management of osteoarthritis has attracted considerable attention; it is one of the five nonvitamin, nonmineral supplements most frequently used by US adults (1). The US FDA currently regulates dietary supplements to ensure that they are produced under cGMP (2).

Urea is commonly used in protein purification, including large-scale purification of recombinant proteins for commercial purposes, and in recombinant protein manufacturing to denature and solubilize proteins (1). In aqueous solutions, urea degrades to cyanate and ammonium, with the maximum degradation rate occurring at neutral pHs commonly used in biological buffers (2). Cyanate is problematic in urea solutions because it carbamylates proteins, which causes unwanted modifications that can alter the protein's stability, function, and efficiency. Therefore, an accurate, sensitive method for determining cyanate in urea-containing buffers is required.

Amylose is an occasionally-branched biopolymer and, together with amylopectin, the hyper-branched component, a constituent of starch. Determination of branching in amylopectin on the basis of amyloses may be performed with the help of synthetic amyloses. Synthetic amyloses from enzymatic (phosphorolytic) reaction were checked for their linearity.

Use imaging mass spectrometry to determine the biodistribution of erlotinib (Tarceva®) in pancreatic tumors from treated and untreated mice.

Supercritical Fluid Chromatography (SFC) is quickly becoming a popular choice by chromatographers for analytical and preparative separations.

By William Goodman, PerkinElmer, Inc.

The analysis of polar compounds in support of clinical and pre-clinical pharmacokinetic studies requires an analytical methodology capable of achieving ultra-low detection and quantification limits. The high sensitivity afforded by coupling HPLC with tandem mass spectrometry (MS–MS) has made it the technique of choice in this environment, but it is subject to the following limitations when reversed phase liquid chromatography (RPLC) is used