Introduction to SPME (Solid Phase MicroExtraction)

Geminal dicationic and polyionic ionic liquids have been prepared for use as stationary phases in capillary gas chromatography. These materials are known to provide higher thermal stability for gas chromatography, broader liquid working ranges and broader selectivity ranges than monocationic ionic liquids and polymeric based stationary phases with similar polarity. Recently, a new ionic liquid stationary phase has been developed that provides a selectivity very similar to a polyethylene glycol (PEG) selectivity but with increased thermal stability and lower bleed. We will compare and contrast the similar but unique selectivity of this new phase with traditional PEG phases using a wide variety of different sample types. We will demonstrate the improved thermal stability and lower bleed through a series of different studies and applications.

Core-type particles are competing strongly with small porous particles to improve the speed and resolution of HPLC and UHPLC experiments. The pioneering Ascentis Express column with Fused-Core® 2.7µm particles has exploded in popularity because it operates more ruggedly at much lower pressure than current sub-2µm porous particles, yet delivers the same ultra-high performance. This unique performance has been largely attributed to very narrow particle size distribution. Fused-Core® design advantages have also become popular for LC-MS because Ascentis Express columns surpass performance of columns with 3µm porous particles and operate ruggedly at higher velocities and similar pressures.

With new, narrow-distribution 5µm Fused-Core® particles, the same design advantages can now be realized over traditional 5µm and 3µm porous particles that remain very popular for HPLC columns. An Ascentis Express 5µm column brings 3µm performance and extreme ruggedness at 5µm pressures to your laboratory. Like Ascentis Express 2.7µm particles, the 5µm particles show flatter van Deemter plots than same-size porous particles and allow separation speed to be maximized with minimal loss of resolution. Extremely high plates per pressure are observed. The core-type 5µm design should replace porous 5µm columns in routine HPLC applications with traditional instruments, and should also compete with porous 3µm columns in many LC-MS applications. Performance will be compared to 5µm and 3µm particle columns, and examples of method transfer will be shown. Ascentis Express 5µm will be available in the same phase modifications as the original Ascentis Express 2.7µm ultra-high performance column. Highly stable columns are available in various IDs and lengths up to 25cm.

Hydrophilic interaction liquid chromatography (HILIC), especially in conjunction with mass spectrometry (MS), has become a powerful tool for the analysis of a wide variety of challenging analytes. Applications of the technique have increased dramatically over the past decade, especially for the analysis of polar analytes where reversed-phase chromatography suffers. HILIC conditions employ a high percentage of acetonitrile which enables facilitated solvent evaporation in LC/MS sources and thus often an increase in analyte response when compared to more aqueous based systems. The increased retention of polar analytes afforded by HILIC provides improved selectivity and decreases the impact of endogenous species, often leading to improved qualitative and quantitative analyses.

Although HILIC has proven useful, it has also been thwarted with complications including difficulties in method development and method robustness.

In this presentation, studies investigating the underlying retention mechanisms dominant in HILIC chromatography are presented and discussed. Along with reversed-partitioning HILIC is well known to exhibit, ion-exchange and the interplay of the dominant mechanisms are unveiled and used to develop a model of overall retention and selectivity. Interactions that operate using different stationary phase chemistries and conditions are presented. The impact of analyte polarity and charge as well as the variations caused by high percentages of organic on these physiochemical parameters are highlighted. Throughout the discussion, examples of use and misuse of HILIC are employed to illustrate these important concepts to build a solid fundamental foundation for efficient and effective use of this powerful technique.

Whether your laboratory uses passive sampling, or active air sampling, this webinar will run through the basics of air monitoring, giving some advice on the best equipment to use for your laboratories application.

Traceable reference materials. RTC have been producing environmental laboratory Proficiency Testing programs and certified reference materials for more than 20 years. More than 20,000 PT samples a year are sent to over 2,500 worldwide participants analysing soil, sediments and both waste and drinking water. This webinar will focus on how Sigma-Aldrich-RTC can support your laboratory with its PT and CRM needs.

The fatty acid composition of a fat or oil is most commonly assessed by gas chromatography, following conversion of the fatty acids methyl esters. The current most refined analytical methods for the quantitation of trans fatty acids rely on the separations provided by long cyanopropyl siloxane capillary columns. The introduction of capillary columns coated with ionic liquids, such as Supelco SLB-IL111, provide an alternative separation tool characterized by a higher stationary phase polarity and selectivity toward geometric and positional isomers of unsaturated fatty acids. The use of these novel capillary columns can provide more refined separations of complex lipid samples. As a result, most conjugated linoleic acid isomers (including t7,c9- and c9,t11-18:1 FA) can be quantitated in a single separation using a 100 m SLB-IL111 capillary column and most 18:1 FA positional and geometric isomers can be separated using a 200 m SLB-IL111. Ionic liquid columns provide more detailed FA profiles, especially for unsaturated fatty acid positional/geometric isomers.

As a leading authority on Karl Fischer Titration, we have extensive experience of product development and customer applications.
Karl Fischer Titration is generally the most accepted method of water content determination and the webinar will provide an introduction to the technique.

Bio-Solid-phase microextraction (BioSPME) is a simple, fast and sensitive non-exhaustive sample preparation technique that allows the integration of sampling and sample preparation steps. The objective of current research was to automate SPME in 96-well plate format for the first time. The proposed system allows sample preparation of >1000 samples/day, simultaneous determination of both free and total concentration and suitability for performing ligand-receptor binding studies. Open-bed configuration of SPME enables direct handling of heterogeneous matrices such as whole blood, thus further simplifying the entire sample preparation process. The automation of SPME in 96-well plate format enables the highest throughput of any SPME technique to date.
In metabolomics studies of biofluids, the efficiency of metabolism quenching and stability of analytes in selected biofluid dictate how accurately the analytical results represent true metabolome composition at the time of sampling. However, complete quenching of metabolism is not easily accomplished and/or changes due to poorly stable compounds can occur, so the processes of sampling and sample preparation can significantly affect metabolome’s composition. The use of SPME for direct in vivo sampling of drugs and metabolites in the bloodstream of freely moving animals eliminates the need for blood withdrawal in order to generate pharmacokinetic (PK) or metabolomic profiles in support of pharmaceutical drug discovery studies. This is particularly important for situations with a limited blood volume such as mice because it enables the use of a single animal to construct an entire profile or conduct longitudinal studies. The aim of the current research was to apply SPME for in vivo sampling in mice for the first time. Furthermore, we investigate the use of in vivo SPME as an effective sample preparation method for both targeted pharmacokinetic and untargeted LC-MS metabolomics studies.

SPME (Solid Phase MicroExtraction) is a solventless extraction technique, used to remove and preconcentrate low levels of volatile and semivolatile organic compounds from soil, water and air.
This webcast will cover the basics of SPME; how to get started, and suggested applications of the technology.

Ionic liquids (ILs) and their components have had a substantial impact on the capabilities and performance of gas chromatography, ESI-MS and MALDI-MS. For GC this ranges from the analysis of complex mixtures by GC x GC, water quantitation and food/beverage analysis among other areas. In MS the most sensitive analysis of anions, phospholipids and metal cations is often done by ESI-MS using complex IL cationic reagents. Also the best MALDI matrices for many proteins, peptides, carbohydrates and synthetic polymers are based on ILs. These will be discussed as time permits.

10 minute introduction to the Ascentis Express family with an emphasis on the benefits of the new ES-Cyano phase. The presentation will then feature a 20 minute advanced level presentation on the molecular interactions contributing to alternative retention and selectivity using Cyano stationary phases

"With a BSc in Biochemistry and Molecular Biology from the University of Western Australia, I started my postgraduate career in the world of cell signaling and transgenic research at the Laboratories of Cancer Medicine at Royal Perth Hospital. After a number of years I relocated to the UK and started working in clinical diagnostic laboratories where I had a change of direction in my career. Moving into the development of HPLC and LC-MS/MS methods for clinical diagnostic analysis, I plied my trade at Guys Hospital and King’s College Hospital’s in London for 6 years before taking my current post at Addenbrooke’s hospital in Cambridge in late 2009. My work interests include new born screening, biogenic amines, steroids and vitamin analysis in an array of human sample types."

Analytical R&D teams are thought of as a core facility within a company. On top of their regular R&D duties, they are often asked to solve production problems, ranging from identifying a contaminant to investigating a competitor’s new product. Learning how to approach these challenges and considering the use of resources beyond your laboratory can save both time and money.

Three case studies will be presented. The case studies involve method development for sub-ppm detection, identifying an off-odor in food, and the detection of a low level pigment. A focus on the specific analytical technology will be presented in the case studies.

Increasing case loads and budget and staffing cuts in forensic laboratories continue to motivate the development of higher throughput methods, particularly for confirmatory analysis of regulated intoxicants. In this work, we have focused on the development of rapid LC/MS/MS methods for the determination of nine opiates including two glucuronide metabolites, and 16 benzodiazepines, including two amino- metabolites. Here we aim to analyze both the parent compounds and important polar metabolites in a single analysis. To this end we have compared the retention of the target compounds on two different reversed-phase HPLC stationary phases: a conventional C18 type phase, and a perfluorinated phenyl (PFP or F5) phase built upon the increasingly popular Fused-Core particle morphology. We see that the F5 phase not only generally exhibits higher retention than the C18 type phase, but also exhibits very different selectivity such that the nine opiates can be nearly completely resolved in under four minutes. We find that the mixture of 16 benzodiazepines cannot be completely resolved in a reasonable (i.e., less than 20 min.) time, however we have developed a separation with no more than three overlapping peaks in an analysis time of five minutes.

Full Title
DEVELOPMENT OF RAPID LC/MS/MS-BASED METHODS FOR CONFIRMATORY ANALYSIS OF OPIATES AND BENZODIAZEPINES

Authors
SPENCER BONNERUP, D. CHRISTOPHER HARMES, TOMAS LISKUTIN, JONNA BERRY, AND DWIGHT R. STOLL

Department of Chemistry
Gustavus Adolphus College
800 West College Avenue
St. Peter, MN 56082

Silica is a popular option for use in “HILIC” mode, but developing new methods can be difficult because the retention patterns are so different from C18. This presentation will provide some practical guidelines for using silica in a HILIC mode – with buffer/acetonitrile mobile phases. Some compound classes are retained under these conditions, and some are not. We will discuss which compounds are good candidates for this system, and summarize how to get started with your method development experiments.

You will learn:
•What compounds can be separated on silica in HILIC mode, and what compounds cannot be separated.
•Starting points for separations, and suggestions for how to adjust conditions for your separation problem.
•Advantages and limitations of silica in this mode.

A comprehensive two-dimensional liquid chromatograph (LC × LC) was constructed from commercially available conventional HPLC equipments. This system utilizes two independently configurable 2nd dimension binary pumping systems to deliver independent flow rates, gradient profiles and mobile phase compositions to dual Fused-Core secondary columns. Very fast gradient separations (30 seconds total cycle time) were achieved at ambient temperature without excessive backpressure and without compromising optimal 1st dimension sampling rates by using superficially porous stationary phases. A practical approach to optimize the various inter-related instrumental parameters will also be presented.

With recent advances in HPLC columns and LC/MS hardware, it is possible to increase the throughput of bioanalytical assays without sacrificing quality. By using fused-core columns, it is possible to decrease the run time from ~4 min to 1 min or less, without the use of UHPLC hardware. High quality methods at high flow rates (1-3 mL/min) using non-ballistic gradients as short as 20 seconds were developed that provide comparable or better performance for accuracy, precision, sensitivity, and specificity than traditional slower LC methods. Data will be presented that show that these assays meet regulatory requirements for bioanalytical work. Limitations in the ultimate speed possible for these assays will also be discussed.

HPLC columns featuring 2.7 µm Fused-Core (superficially porous) particles with 90 Å pores demonstrate very fast separations of small molecules because of high efficiency and a flat van Deemter plot. These particles rival the efficiency of sub-2 µm totally porous UHPLC particles, but show only about one-half the backpressure. Fused-Core 2.7 µm particles with wider (160 Å) pores have been optimized for the rapid separation of peptides and small proteins. The higher efficiency and lower pressure drop of Fused-Core particles allows preparation of longer columns with very large numbers of theoretical plates. This dramatically increases the peak capacity of the column system, which facilitates qualitative and quantitative HPLC and LC-MS analysis.

15 minute introduction to the Ascentis Express family with an emphasis on the benefits of the new F5 phase. The presentation will then feature a 45 minute advanced level presentation on the molecular interactions contributing to alternative retention and selectivity using F5 (pentafluorophenyl) stationary phases