Application Notes

Semivolatile analyses using methods similar to US EPA method 8270 (1) are important in environmental laboratories worldwide. A number of acidic compounds such as benzoic acid or 2,4-dinitrophenol and strong bases such as pyridine or benzidine are active species found in the semivolatile sample set. These highly polar species are particularly susceptible to adsorption into active surfaces in the sample flow path, including the column itself. System and column inertness are critical for effective analysis of these active chemical species.

Cefepime is a fourth generation cephalosporin (1). During preparation and storage, cefepime degrades by release of the N-methylpyrrolidine (NMP) side chain and opening of the beta-lactam ring. An NMP concentration increase will directly affect the potency of the active component of the drug. Therefore, it is critical to determine the amount of NMP in cefepime. The US Pharmacopeia (USP) monograph specifies the limit of NMP to <0.3% in cefepime hydrochloride and <1% in cefepime for injection (2,3). The latter is a dry mixture of cefepime hydrochloride and L-arginine. The current USP method uses cation-exchange chromatography with non-suppressed conductivity detection to determine the limit of NMP in cefepime. There are several disadvantages to this method, such as the ~3-4 h time required per injection, a lack of retention time stability for NMP in standard and sample solutions, and a lack of sensitivity. In this paper, we describe an improved method using a hydrophilic, carboxylate-functionalized cation..

In general, polymer-based columns have a broad pH range (pH 2 to 13), and some have high temperature tolerance (up to 150°C or higher). Considerably large selectivity changes can be obtained by varying analysis temperature and mobile phase pH. Having control on these two parameters over wide ranges can be especially useful in method development.

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

Political priorities as well as economic interests have fueled a dramatic growth in the biofuel industry, due much in part to research funding and tax incentives. Currently the world's ethanol production is estimated to be over 16 billion gallons a year. This number is expected to increase in the next few years, reaching an estimated yearly production of 20 billion gallons by 2012. Today, many ethanol producers add fermentors to expand their production capacity. In order to continue using existing HPLC equipment for the increased monitoring, increased analytical throughput is needed.

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.

Use of a modified QuEChERS sample preparation procedure was fully evaluated for identification and quantitation of pesticides in lettuce using gas chromatography and ion trap mass spectrometry. Final results compared favorably to the reporting and detection limits offered by several worldwide agencies, demonstrating a robust and reliable solution for analyzing pesticide residues in lettuce.

The second unregulated contaminant monitoring regulation (UCMR2) program was developed to monitor US drinking water sources for currently unregulated compounds. EPA Method 527 is categorized under List 1; Assessment Monitoring in the UCMR2 program. EPA Method 527 focuses on a wide range of semi volatile organic contaminants, including pesticides that were deferred during the first UCMR, flame retardants, and pyrethroid pesticides. This application for EPA Method 527 employs SPE with analysis by gas chromatography–mass spectrometry (GC–MS).

Paracetamol is a major ingredient in numerous medications due to its analgesic and antipyretic properties. During its synthesis (Figure 1), a total of ten process-related impurities are observed. Several HPLC applications have been developed for the monitoring of these impurities (1, 2), including the European Pharmacopoeia which has adopted an isocratic HPLC method using a silica-based C8 column with 5 μm particle size, requiring a run time of 45 min (3). By using a gradient method and standard HPLC instrumentation, the analysis can be reduced to 7 min (4).

A large percentage of commercial and investigational pharmaceutical compounds are enantiomers and many of them show significant enantioselective differences in their pharmacokinetics and pharmacodynamics. The importance of chirality of drugs has been increasingly recognized, and the consequences of using them as racemates or as enantiomers have been frequently discussed in the pharmaceutical literature during recent years. With increasing evidence of problems related to stereoselectivity in drug action, enantioselective analysis by chromatographic methods has become the focus of intensive research of separation scientists. Most of the pharmaceutical and pharmacological studies of stereoselectivity of chiral drugs before the mid eighties involved pre-column derivatization of the enantiomers with chiral reagents forming diastereomers.

Traditionally the analysis of water-soluble vitamins by reversed-phase HPLC has been complicated by the lack of retention for these compounds on conventional silica C18 columns. Here we demonstrate efficient, multi-mode, baseline resolution of six water-soluble vitamins in 6 min using a ZirChrom®-SAX column.

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.

The analysis of polar compounds in support of clinical and preclinical 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

Capillary flow technology (CFT) devices are microfluidic components that extend capillary GC capabilities through simple and robust connections between pressure/flow modules and columns. One of the most powerful and simple is the CFT Tee. This is especially useful in GC–MS analysis providing (1) rapid column and inlet maintenance without MSD venting and (2) the capability of rapidly removing late eluting interferences from the column by forcing their retreat into the injection port through "backflushing". Removing these interferences improves column and detector longevity and analytical integrity. Backflushing is very valuable for trace GC–MS analysis in samples from complex matrices like soil, foods or tissues. The CFT Tee uses pressure-pulsed injections and constant flow mode with minimal loss in the MS signal. This approach will be useful to all GC–MS users who want to improve their instrument uptime.

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

A new APCI/APLI source enables a high resolution TOF-MS to be coupled on LC and GC, thus increasing flexibility and performance of TOF-MS in combination with GC.

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.

Under the registration, evaluation and authorization of chemicals (REACH) Regulations (EG) no. 1907/2006 the producer, manufacturer or importer of chemical substances/formulations have to register their products (more than 1 ton/year) at the chemical agency.

PEGylation, the process by which polyethylene glycol (PEG) chains are attached to protein and peptide drugs is a common practice in the development of biopharmaceuticals to prolong serum half-life and improve pharmacokinetics of a drug. There is increasing demand for chromatographic methods to separate the modified isoforms from the native protein. This application note describes the use of size exclusion and ion exchange chromatography for the characterization of PEGylated lysozyme.

In March 2007, several North American manufacturers of pet food voluntarily issued nationwide recall notices for some of their products that were reportedly associated with renal failure in pets. The raw material wheat gluten, used to manufacture the pet food, was imported from China and was identified as the source of contamination.

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

Guest Editor Peter Schoenmakers provides an introduction to LCxLC, and then goes on to talk about peak capacity, sample dimensionality, phase orthogonality, and some of the successes of the technique and the obstacles yet to be overcome.

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.

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.

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.