The Application Notebook-06-01-2008

Short analyses time and high resolution are in great demand from R&D and QC departments within the pharmaceutical industry. Sub-two micron ODS reversed phase columns have recently been introduced to meet these requirements, but these columns require an ultra-high pressure HPLC system to achieve optimum performance. TSK-GEL ODS-140HTP, 2.3mm columns from Tosoh Bioscience have been developed to offer a combination of short analyses time and high resolution separations that can be run at modest pressures, making these columns compatible with conventional HPLC instrumentation. The polylayer bonding chemistry of these columns results in highly efficient and physically stable columns when operated at high linear velocities. In addition, TSK-GEL ODS-140HTP, 2.3mm columns can be efficiently operated at pressures not exceeding 9000psi in UPLC® and other ultra-high pressure HPLC systems, as well as in traditional HPLC systems.

Pharmaceutical actives and impurities often contain sulfur in the form of a sulfone or sulfoxide group. Both groups have dipole moments, adding a hydrophilic character to compounds containing these functional groups. The analysis of hydrophilic compounds on traditional alkyl columns (e.g. C18) can be problematic, since alkyl columns depend on hydrophobic interactions for retention. Since the sulfone and sulfoxide groups contain π-π bonds, the biphenyl column's ability to undergo π-π interactions makes it an excellent choice when increased retention or selectivity of compounds containing these groups is desired. To demonstrate the selectivity of the biphenyl phase towards aromatic compounds containing sulfur groups, a set of target compounds was selected and analyzed on C18, phenyl, and biphenyl columns.

Synthetic fused silica capillary tubing has proven to be a vital component in many separation science techniques. Recently, it has expanded to serve an array of scientific markets. In most cases, an understanding of the internal surface chemistry is of fundamental importance.

Mercury pollution mainly originates from industrial activities such as chlorine production, garbage incineration and above all coal-fueled power generation. The US Environmental Protection Agency (US EPA) considers mercury as highly toxic with a pronounced accumulative and persistent character.

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MEPS uses a barrel insert and needle (BIN) device to reduce Solid-Phase Extraction (SPE) to a micro-scale suitable for small volume samples and for the online adaptation of conventional SPE techniques. Because the SPE cartridge (BIN) is incorporated into the needle assembly of a gas-tight syringe, MEPS is also a simple field-portable SPE device that may be operated manually without need for sampling pumps or, alternatively, may be incorporated into robotic samplers. MEPS devices are of glass and stainless steel construction allowing them to be fully immersed for sampling at depth or, alternatively, used at needle depth to avoid perturbing the stream from which the sample was drawn. An extension pole allowed MEPS to be used to sample back along pipes or down inspection vents. When sampling from drainage pits and open sumps, there was minimal requirement to remove grates to gain access. An extension pole also allowed sampling from outflows that were offensive and could be readily adapted for safe sampling of..

his application note describes the extraction of a variety of basic drugs from plasma using EVOLUTE CX mixed-mode resin-based SPE. The analyte suite includes basic drugs with wide ranging pKa and logP values.

There are many misconceptions about what it means to perform fast gas chromatography (GC) and what the term fast GC implies. Fast GC is often associated with the use of hydrogen as a carrier gas and, although this is certainly a good approach, it is not always necessary to shorten the analysis time. A second misconception is that changing column dimension results in time-consuming method development. Using high-efficiency GC columns can greatly reduce the analysis time and when coupled with the method translation software, the time spent on method development can be greatly minimized.

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Viscotek has been a strong advocate of good chromatography as a prerequisite for GPC (Gel Permeation Chromatography) data accuracy. Our recent work in application development has been driven by extremely difficult samples from industrial, biopharmaceutical, and academic sources. These samples present challenges ranging from sample solubility, column adsorption as well as detection issues. This report will attempt to highlight a new approach that could be very helpful in certain advanced GPC applications.

EPA Method 525.2 describes the procedure to determine low ppb levels of semi-volatile organic material in drinking water using solid phase extraction (SPE) or liquid-solid extraction (LSE) techniques. The City of Fort Worth, Water Department implemented an automated SPE process for the analysis of semi-volatiles by EPA Method 525.2, using the Atlantic "Certified for Automation" SPE Disk for EPA Method 525.2. Ethyl acetate, methanol, and water were used to condition the Atlantic disk prior to the extraction step. The extraction solvents used were a 1:1 mixture of methylene chloride and ethyle acetate. Extracts were then analyzed by GC–MS using a splitless injection technique.

In pharmaceutical development, it is important to analyze small molecules or their metabolites in biological fluids. For this purpose, the analytical methods such as sample pretreatment, 2D-LC and LC–MS have been developed. However there are still problems of resolution and protein adsorption. As a result, satisfying analytical results have not always been achieved.

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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.

With the forthcoming USP monograph <1058>, many laboratories are in the process of reexamining their high performance liquid chromatography (HPLC) instrumentation qualification practices. This article demystifies the qualification procedures and proposes a well designed, easy and simple set of experiments upon which to establish internal standard operating procedures (SOPs) for the complete qualification of HPLC instruments. A key concept is the development of a consistent test system, comprised of premade test solutions, a prequalified HPLC column, standardized protocols, and validated software that can be prepared in-house or purchased commercially as a kit. This system can be applied to any HPLC system worldwide, to produce comparable test results under uniform conditions. The test system is designed to be rapid, with a comprehensive performance qualification being completed in about 2 h for isocratic, and 3 h for quaternary gradient systems.

Two of the most commonly occurring unpleasant odor-causing compounds in drinking water are geosmin and 2-methylisoborneol (MIB) (Figure 1). Geosmin is produced primarily by blue-green algae (cyanobacteria) and actinomycete bacteria, and MIB is produced by certain species of cyanobacteria, primarily Oscillatoria. Many environmental laboratories are required to detect these compounds as low as 1–3 ppt concentration and measure quantitatively at 5 ppt.

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Dionex has developed a new standard for flow-through solvent extraction which allows accelerated solvent extraction (ASE®) of matrices that have undergone acid or alkaline pretreatment or digestion. The new ASE 150 and ASE 350 systems use extraction cells and post-cell solvent pathways constructed of Dionium™ material. This pH-hardened substance resists corrosion under acidic or alkaline conditions used in standard pretreatments, widening the scope of ASE applications and significantly expanding its capabilities.

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Several common birth control formulations contain both drospirenone and ethinyl estradiol. A highly selective and sensitive analytical method for the analysis of drospirenone in human plasma has been developed for use in bioequivalence studies. The solid-phase extraction (SPE) and UPLC®–MS–MS methodologies are described as well as performance against validation parameters.

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.

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The purge-and-trap (P&T) technique for analysis of volatile organic compounds (VOCs) was pioneered in the 1970s at the United States Environmental Protection Agency (USEPA) research laboratory in Cincinnati. Many of the operational parameters developed during this time period are still included in USEPA methods. While these parameters still produce good analytical results, they do not take advantage of advances in instrumentation that enable analysis of emerging contaminants such as fuel oxygenates, and increased sample throughput.

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H-SRM provides excellent selectivity for accurate identification and quantification of pesticides in matrix, demonstrating high productivity for effective control at international maximum residue levels (MRLs).

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Bradykinin, a 9 amino acid peptide, is a physiologically and pharmacologically active peptide of the kinin group of proteins, which is used in the development of antagonists and therapies for hereditary angioedema. In this application, 50 mg of crude bradykinin, synthesised on a StratoSpheresâ„¢ PL-Rink resin, is purified using an HPLC method that can be scaled from the laboratory through to full production.

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The United States Geological Survey (USGS) has found pharmaceuticals and personal-care products (PPCPs) containing known or suspected endocrine-disruptors in U.S. rivers. As such, it is important to use adequate techniques to help identify these compounds and possible metabolites.

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With recent advances in technology and the use of ultra high pressure chromatography systems becoming more commonplace, it is of use to compare reproducibility of retention time and peak area of UHPLC vs HPLC.

Azo dyes are used widely in the manufacture of various consumer goods such as leather, textiles, plastics, paper, hair care products, and cosmetics. On September 11, 2003, the European Union enacted European Parliament Directive 2002/61/EC, prohibiting the manufacture and sale of consumer goods containing specified azo dyes (1). The azo dyes of concern are those that can be reduced to aromatic amines. There are 22 aromatic amines classified as carcinogenic or potentially carcinogenic to humans.

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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.

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Aminoglycoside antibiotics are among those commonly used in animal feeds to manage intestinal microorganisms. The beneficial effects include improved growth and generally healthier animal populations. Use of antibiotics creates a demand for analytical procedures to verify concentrations in pre-mixes and feeds and in some instances for residue analysis in animal products.

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Food and Beverage

June 01, 2008

Mycotoxins, toxic secondary metabolites of several fungal species, represent food safety issues of high concern. Deoxynivalenol, the most abundant trichothecene mycotoxin, can be found worldwide as a contaminant of wheat, barley, maize and other cereals (1,2). The transmission of deoxynivalenol from barley into beer has been reported in several studies (3,4). Therefore, its levels should be controlled.

Pharmaceutical
The Application Notebook

June 01, 2008

A new phenylhexyl bonded phase provides an alternative selectivity for optimizing HPLC method. This also applies when different types of phenyl bonded phases are compared.

Supercritical fluid chromatography (SFC) is a normal phase separation technique. In SFC, supercritical CO2 in combination with one or more polar organic solvents, most commonly alcohols, is used as the mobile phase. Owing to the lack of intermolecular interactions, supercritical fluid typically possesses lower viscosity and higher diffusivity than those solvents used in traditional high performance liquid chromatography (HPLC). This allows for higher flow rates, faster analyses, and the use of longer columns for higher chromatographic efficiencies. Initially deemed a niche chromatographic technique for chiral separation, the horizon of SFC applications has rapidly expanded to include achiral analyses of natural products, biodiesel, oligomer, pesticides/herbicides, and peptides. This is due, in part, to the improvements in detection choices and performances for SFC. Evaporative light scattering detectors (ELSDs) coupled with SFC have found wide use in many pharmaceutical and chemical laboratories (1).

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This application note describes a fast and sensitive LC-MS method using a Hypersil GOLDâ„¢ column on a Thermo Scientific LC-MS system for the quantitative analysis of two widespread PFCs, perfluorooctanoic acid (PFOA) and perfluorooctansulfonate (PFOS).

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

The Application Notebook

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.