The Application Notebook-06-01-2009

The analysis of anions and cations is critical during quality control. In this example, all major ionic components in mineral water are identified and quantified in a single run, with no sample preparation or concentration step.

The Application Notebook
Food and Beverage

June 01, 2009

The screening of pesticides, mycotoxins, and veterinary drugs is of great importance in regulated environments such as food or feed analysis. Due to some of the limitations of traditional triple quadrupole approaches (for example, targeted analyte detection, limited number of compounds, and unidentified unknown compounds), there is currently a trend towards use of full-scan MS data for the analysis of residue samples. Current screening approaches mainly rely on the use of ToF instruments coupled to U-HPLC delivering mass accuracy (~5 ppm) at a maximum resolution of <15,000. This can produce inaccurate mass measurements due the presence of unresolved background matrix interferences. In this work we show a full-scan MS screening approach with the Thermo Scientific Exactive mass spectrometer, a novel single-stage Orbitrapâ„¢ MS capable of providing precise mass accuracies at resolutions of up to 100,000 without the need for internal mass calibration.

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It is no secret that these are tumultuous times we live in. With the economic slowdown, increased dangers abroad, and the recent string of scares involving toxins in everyday foods, there is no shortage of things to worry about in today's world. At LCGC, we've always felt that our Application Notebook is a reflection of not just the recent developments in chromatography and the field of separation science in general, but the world as a whole.

Since melamine and its metabolites are extremely polar compounds, they serve as very good candidates for HILIC chromatography. Simultaneous and fast determination and confirmation of melamine and cyanuric acid along with two other compounds using a novel amino bonded phase in HILIC mode coupled with a complete solutions approach is presented.

Melamine is an organic base and a trimer of cyanamide, with a 1,3,5-triazine skeleton. Melamine can react with formaldehyde to produce melamine resin, a very durable thermosetting plastic, and melamine foam, a polymeric cleaning product. Some end products made from melamine include countertops, dry erase boards, fabrics, glues, housewares, and flame retardants. Melamine is also one of the major components in Pigment Yellow 150, a colorant in inks and plastics.

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An Agilent ZORBAX Rapid Resolution HT Eclipse Plus Phenyl-Hexyl column resolved many compounds from Stevia rebaudiana Bertoni plant extracts using an isocratic reversed phase method with UV and MS detection instead of a typical approach employing an amino column with a refractive index or other special detector. The Eclipse Plus Phenyl-Hexyl method's advantages include high peak capacity, isocratic mobile phase, low acetonitrile consumption, and MS compatibility.

The United States Food and Drug Administration (U.S. FDA) and the European Union (EU) have published regulations and provided guidance on the use of chemicals in food packaging materials. Tekmar provides method parameters for the HT3â„¢ Automated Headspace Analyzer for the determination of VOCs that may be contained in food contact packaging materials under various conditions. Temperature and mixing parameters were investigated to determine which VOCs may be released when subjected to these conditions.

Simulated Moving Bed technology (SMB) is a continuous chromatography technique widely used in the pharmaceutical/biotechnology industry. The continuous chromatographic separation of sodium sulphate (Na2 SO4) from a mixture of glucose and mannose was established by using the Orochem Zuccheroâ„¢ columns and the SMB technology. The main advantages of SMB over batch chromatography includes better yields, higher purity, and decreased solvent usage, which together make the SMB technology economically viable and ideal for desalting applications.

Based on the QuEChERS AOAC Official Method 2007.01, sixteen pesticides were used for evaluating the performance of the Agilent's AOAC Buffered Extraction and SampliQ QuEChERS Dispersive SPE kits for General Fruits and Vegetables. Apple was selected as the fruit matrix. The pesticides are from the "representative pesticides" list (1). A method working well for these representative pesticides should work equally well for the other pesticides that are routinely monitored in multi-class, multi-residue methods. The compounds are from nine different pesticide classes: including acidic, basic, neutral, base-sensitive, and acid-labile pesticides. The selected pesticides are suitable for LC–MS-MS analysis. The MRLs of these pesticides have been set at 10 ng/g or higher (2–4).

Homeland security requirements create the need for portable mass spectrometers, with ion detectors capable of performing at elevated pressure.

Many polar compounds are difficult to retain by reversed-phase analysis without the use of ion-pair chromatography. We have overcome this disadvantage with a revolutionary phase structure called "Multi-mode ODS." This technology uses uniformly blended packing material consisting of two types of porous silica particles: 3 μm silica substituted with ODS+cation ligands and 3 μm silica substituted with ODS+anion ligands (Figure 1). This novel multi-mode ODS column, named Scherzo SM-C18, enables a multi-separation mode, consisting of: anion exchange, cation exchange, normal phase, and reversed-phase. In this article, we will validate this multi-separation mode by separating compounds which are difficult to separate on ODS column.

Polybrominated diphenyl ethers (PBDEs) are bioaccumulativetoxic compounds that are used as flame retardants. They have high boiling points and low thermal stability which makes analysis by GC–MS challenging. Additional bromines increase the thermal instability. A method was developed to analyze for polybrominated biphenyls using a large volume injection, short analytical column, and high mass tuning algorithm on the Thermo Scientific DSQ II with detection by EI Single Ion Monitoring (SIM).

Tobacco Specific Nitrosamines (TSNA) are a group of carcinogens found only in tobacco products and are formed from nicotine and related alkaloids during the production and processing of tobacco and tobacco products (1). Due to their carcinogenic properties, efforts have been made to reduce TSNA levels in tobacco products. The project goal was to demonstrate a high throughput and sensitive method to monitor TSNA levels.

In the past few decades, single enantiomers and stereoisomers have overtaken achiral molecules in the percentage of approved drugs in the market. Because isomers can have different biological/pharmacological/toxicological properties, authorities, such as the European Pharmacopoeia and the FDA, have asserted escalated emphasis on controlling isomer content in drug compounds and require that stereoisomers, "be treated as separate drugs and developed accordingly" with rare exception. The separation and quantification of stereoisomers is therefore of great importance, especially when considering pharmaceutical compounds (1).

The Application Notebook
Biological

June 01, 2009

Viruses are packets of infectious nucleic acid (either DNA or RNA) surrounded by a protective coat consisting of a large number of protein subunits. Since viruses can cause various diseases - some life-threatening - characterizing virus particles thoroughly in terms of their size distribution, aggregation, and absolute counts-per-unit volume is of extreme importance.

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This article describes the use of combined ion chromatography-mass spectrometry (IC–MS) and ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP–MS) to analyze potentially harmful compounds.

Trace-level chlorinated hydrocarbon analyses using methods such as U.S. EPA Method 551.1 are important tools for assessing organochlorine contamination in water. The wide diversity of target organochlorine compounds can prove chromatographically challenging due mainly to their high volatility and limited retention. This application note shows the benefits of using an Agilent J&W HP-1ms Ultra Inert Capillary GC column as the primary column for detection in this dual-column analysis.

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Biodiesel is much in the news today as an alternate fuel source that is safe and nontoxic. It is renewable, via farming and recycling, and is biodegradable. It is cleaner burning than petroleum-based gasolines, with virtually no sulfur and with no net carbon load to the atmosphere.

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Human health not only depends on providing good medical care, but also on the priority given to prevent exposure to environmental and other health risks. Persistent Organic Pollutants (POPs) are organic compounds typically of anthropogenic origin that resist degradation and accumulate in the food chain and are associated with adverse effects on human health and the environment (1). Due to their toxicity to humans, at much lower concentration than other pollutants, it is important to monitor compounds like polychlorinated dioxins/furans PCDD/Fs, DLPCBs, BDEs, and PCNs. More sophisticated requirements are needed for their analysis. In the past, extraction and clean-up of POPs present in fish and biota samples were conducted with procedures such as Soxhlet extraction, acid digestion, and liquid-liquid extraction. The clean-up of these samples was accomplished through chromatographic columns using different types of adsorption media such as silica, alumina, and carbon. These analytical methods used for analysis..

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Although supercritical fluid chromatography (SFC) is generally regarded as a technique to separate enantiomers, it has also been proven in many laboratories as a fast approach for the purification of impurities in achiral samples.

Rapid sample preparation using the CUSTODIONâ„¢ solid phase microextraction (SPME) syringe was applied to chemical warfare agents (CWAs), CWA simulants, by-products, and precursors. The samples were analyzed quickly and reliably with a sample-to-sample cycle time of less than 3 min using the GUARDIONâ„¢-7 portable capillary gas chromatograph toroidal ion trap mass spectrometer (GC–TMS).

The Application Notebook
Biological

June 01, 2009

Over-expression of recombinant proteins is commonly used for the production of protein reagents in industry and academia. Problems often occur relating to the stress put on the cells to deal with this huge increase in synthesis. Cellular proteins that are part of the protein synthesis machinery are often up-regulated under such conditions. Large quantities of the recombinant protein can be bound to these cellular proteins, making purification difficult.

Protein phosphorylation is one of the most prevalent intracellular protein modifications, regulating numerous cellular processes including cell differentiation, proliferation, and migration. Approximately 30% of cell proteins are phosphorylated at any given time and changes in protein phosphorylation often signal developmental or pathological disorders (1). To better understand the role of protein phosphorylation, it is important to separate the phosphorylated forms of a given protein.

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The new ultrafleXtremeâ„¢ exceeds all current expectations of MALDI-TOF/TOF technology: A proprietary kHz smartbeam-IIâ„¢ MALDI laser integrated with a novel FlashDetectorâ„¢ and re-engineerd electronics makes it the only MALDI-TOF/TOF on the market to provide kHz acquisition in MS and MS-MS modes. It generates a new level of data quality in applications such as LC-MALDI proteomics, high resolution tissue imaging based biomarker discovery or Top-Down Sequencing.

In U.S. EPA Method 5035 there are two collection options for samples containing high levels of volatile organic compounds (VOCs). The first involves collecting a bulk soil sample in the field. In the lab, the bulk soil is dispersed in a water miscible solvent. Next, an aliquot of the solution is added to 5 mL of water and finally, the sample is purged. The second option is to collect a 5 g soil sample and add it to a pre-weighed vial containing a prescribed amount of water miscible solvent. An aliquot is then purged. Both of these processes are time consuming and have the potential to introduce human error both in the field and in the laboratory.

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Polynuclear aromatic hydrocarbons (PAHs) are carcinogenic condensed ring aromatic compounds widely found as trace pollutants in waters, wastes, air particulates, soil, and foods. PAHs can be monitored routinely using HPLC with a combination of UV and fluorescence detection as prescribed in EPA methods 550.1, 610, and 8310. Conventional HPLC analysis of 19 PAHs typically requires 20 min and uses 25 mL of acetonitrile. However, there is a continual drive to improve productivity and reduce solvent consumption and waste in chemical analysis. Using ultra high pressure LC (UHPLC) with sub-2 μm particle-size columns, we demonstrate a 3-fold improvement in throughput and a 90% reduction of mobile phase solvent in the determination of 19 PAHs.

Most U.S. Environmental Protection Agency (U.S. EPA) methods for analysis of volatile organic compounds (VOCs) specify purging with helium for 11 min at 40 mL/min, making purge-and-trap (P&T) one of the biggest consumers of helium in a laboratory. Compared to helium, nitrogen is abundant, inert, and can be purchased at affordable prices.

Iodine is an essential nutrient in seawater, seafood, and iodine-enriched foods, such as iodized table salt. The most common forms of iodine in the diet are iodide and iodate, with additional iodo-organic compounds providing a small fraction of the bio-available iodine. Iodine deficiency affects thyroid hormone production and leads to developmental diseases, goiter, and paralysis (1).

Polysaccharide-based chiral stationary phases (CSP) are successfully employed to achieve the majority of analytical and preparative enantioselective separations. However, there are a number of separations where significant modification or enhancement of selectivity is necessary. Two new columns, CHIRALPAK AY-H and CHIRALCEL OZ-H, have been introduced to meet these needs.

Evaporation-free extraction (no drying down) is highly desirable because of its reduced cost and pollution, higher speed, and less possibility for contamination and conversion. Presented in this article are four types of evaporation-free extraction that are widely applicable. The first one is for the determination of didanosine over the range of 25.02–2502.00 ng/mL by injecting solid-phase extraction (SPE) eluate directly. The second is for the determination of betamethasone phosphate over the range of 2.51–250.60 ng/mL by injecting SPE eluate after pH adjustment. The third is for the determination of sumatriptan over the range of 0.99–99.40 ng/mL based upon SPE with high organic washing and low organic elution. The fourth is based upon automated dilution after protein precipitation for the determination of raloxifene-4'-glucuronide and raloxifene-6-glucuronide over the ranges of 2.02–202.40 ng/mL and 0.40–39.95 ng/mL, respectively.