Application Notes

In this instalment of "Sample Preparation Perspectives", columnist Ron Majors discusses advanced topics such as multimodal SPE, restricted-access media, molecular imprinted polymers, immunoaffinity extraction phases and other class-or compound-specific sorbents...

Michael D. Buchanan, Sigma-Aldrich/Supelco

Molecularly imprinted polymers (MIPs) are synthetic polymeric materials that mimic immunosorbents. They are widely used as sorbents for solid-phase extraction (SPE). The most common way to synthesize them is bulk polymerization because of its simplicity and versatility. This produces a hard monolith that has to be ground and sieved to obtain particles in the desired size range. However, the partial loss of the materials as fine dusts; the irregular shape of the particles produced and their wide size distribution, have led to a search for different polymerization methods to offset the drawbacks of the bulk polymerization process.

TN012 Sulfanilamides from Honey using strata-X-C phenomenex

Molecularly imprinted polymers (MIPs) are synthetic polymeric materials that mimic immunosorbents. They are widely used as sorbents for solid-phase extraction (SPE). The most common way to synthesize them is bulk polymerization because of its simplicity and versatility. This produces a hard monolith that has to be ground and sieved to obtain particles in the desired size range. However, the partial loss of the materials as fine dusts; the irregular shape of the particles produced and their wide size distribution, have led to a search for different polymerization methods to offset the drawbacks of the bulk polymerization process.

LC–MS–MS methods for the unambiguous identification and quantification of pesticides in complex matrix samples are well known and widely used. Triple quadrupole systems have proven useful for this task because of their high specificity in MS–MS mode and their low detection limits. However, working in targetted MS–MS mode prevents the detection of other compounds.

Very low level detection of mycotoxins in the agriculture products has become increasingly important. Mycotoxins are very resistant fungal metabolites that can remain in foods after processing and, sometimes, even after cooking. They are considered potent carcinogens and can be found in many varieties of foods. Of the mycotoxin class, aflatoxins are considered especially harmful, being both acutely and chronically toxic. Aflatoxin B1 and B2 are some of the most potent hepatocarcinogens known. Along with aflatoxins G1 and G2, even extremely low levels of these aflatoxins in the diet are important public-health concerns. With this in mind, this application note describes a specific, robust HPLC method for the low-ppb detection of aflatoxins B1, B2, G1 and G2 in corn.

Mixed mode chromatography combines aspects of ion exchange chromatography and conventional reversed-phase (RP) chromatography. The combination of both hydrophobic and ion-exchange properties allows for independent control of retention for ionizable and neutral molecules.

UltraPerformance LC (UPLC) has been widely accepted by chromatographers because of improvements over HPLC in the sensitivity, resolution and speed of separations. As scientists begin to use this technology for impurity and metabolite profiling, they will need to transfer the methods to preparative LC to isolate and purify their compounds for further research. Therefore, it is necessary to systematically transfer UPLC assays not only to HPLC, but, more importantly, to preparative chromatography. In this application, we provide information on how to scale a UPLC impurity/degradant separation to a preparative LC separation.

Suppressed conductivity detection is a well-developed method for detecting charged species. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a well developed method of separating substances on the basis of hydrophobicity. There are some situations where it is advantageous to use these two methods together. Perfluoro-acids (PFOAs) are one class of compounds that are ionic, hydrophobic and have low UV absorbance and are, therefore, suited to this combination.

Polycyclic aromatic hydrocarbons (PAHs) are commonly found throughout the environment in soil, water and adsorbed to fine particulate matter in air. Of the 16 common PAHs, 7 have been classified as animal carcinogens by the International Agency for Research on Cancer (IARC). Resulting from this classification, PAHs are monitored and regulated in the environment.

Pesticide contamination of foodstuffs has become a worldwide concern, prompting various levels of regulation and monitoring. Traditionally, pesticides are quantified with gas chromatography (GC) combined with selective detectors (ECD, FID, etc.). Selective GC detectors are great tools to quantify one or two classes at a time. However, screening for a number of different classes of pesticides requires multiple runs utilizing various GC configurations to achieve sufficient chromatographic resolution for unambiguous quantification. Gas chromatography–mass spectrometry (GC–MS) provides positive confirmation of various pesticides in a single analytical run because its superior selectivity allows interference-free quantification even with peak coelution. GC–MS has become a preferred technique for pesticide analysis because of its single-run capability.

The continual increase in sample numbers in busy labs means that it is often difficult for quality control or contract analysis labs to maintain short turnaround times, particularly when instruments are already running at full capacity. To address the need for faster analysis while retaining the quality of separation offered by dedicated amino acid analysers, an improved formulation of sodium citrate based buffers has been developed by Biochrom.

The exploration of myxobacterial metabolite profiles by LC–MS screening for the presence of new natural products is described. Extracts from fermentations of Myxococcus strains are analysed by UPLC-coupled ESI-TOF mass spectrometry and the obtained data are processed using principal component analysis (PCA). The generation of molecular formulae from accurate mass measurements facilitates rapid compound identification.

Brian De Borba and Jeffrey S. Rohrer, Dionex Corporation

Brian De Borba and Jeffrey S. Rohrer, Dionex Corporation

Bob Wiedemer, Grace Davison Discovery Sciences

Xiaodong Liu and Christopher Pohl, Dionex Corporation

Daniel Krug, Gabriela Zurek, Birgit Schneider, Carsten Bässmann and Rolf Müller, Bruker Daltonik

Eric S. Grumbach, Diane M. Diehl and Jeffrey R. Mazzeo, Waters Corporation

William Goodman and Thomas Meaker, PerkinElmer Life and Analytical Sciences

Gas chromatography (GC) coupled to time-of-flight mass spectrometry (TOF-MS) offers unique solutions for various analytical applications including the analysis of food quality, authenticity and safety markers. This article provides a general overview of TOF-MS basic features, highlighting its advantages and limitations compared with GC using conventional mass analysers. Examples of recent results obtained for selected food contaminants and flavour components are described to illustrate the potential of this recently introduced technique.

MIP Technologies has signed an exclusive global distribution agreement with Supelco, a division of Sigma-Aldrich. The agreement is for the distribution of analytical and preparative molecularly imprinted polymer (MIP) separation products.

The analysis of peptides and additives using a new HPLC innovative column technology, Pathfinder, was shown. For the analysis of flavours in lemon juice GC–MS with a new MS library, FFNSC, with linear retention indices was used.

Soft drink formulas often include preservatives, artificial sweeteners, flavours or caffeine in their list of ingredients. Using the Acclaim OA column, as many as eight common additives may be determined in a single run. Many of these additives are hydrophilic organic acids for which this column was designed. Notably, benzoate and sorbate, which do not resolve on C18 columns at low pH, are fully separated.

Accurate mass measurements are a key element of chemical characterization. However, the accepted mass accuracy tolerance of 3–5 ppm can still leave significant ambiguity in the proposed chemical formula. Consequently a further input from other analytical techniques such as NMR or MS/MS, along with some judgment based on the synthetic history is often required to arrive at a confident formula assignment.

Glyphosate [N-(phosphonomethyl) glycine] is a broad spectrum, non-selective herbicide, which acts by inhibiting the shikimic acid pathway in plants. Recent studies have raised global health and environmental concerns about glyphosate's use.1 Glyphosate readily breaks down into aminomethyl phosphonic acid (AMPA) in the environment; requiring accurate measurement. Both highly polar compounds present an analytical challenge to the chromatographer (Figure 1). Typical silica based reversed-phase C18 columns experience difficulty with the retention of such polar compounds, and may generate non-resolved co-eluting peaks, often with polar analytes eluting in the void volume. Traditional analytical methods require complex eluents and time consuming derivatization steps to achieve retention on a reversed-phase support.

Column lifetime is a more and more important issue when developing an analytical method for HPLC. Besides sample treatment, column cleaning and storage, operational parameters of the analytical method will have an influence on column lifetime. This question may not always be addressed early enough in the methods development process.