"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."Read more >
Multiplexed protein assays have tremendous potential in clinical diagnostics, in particular measurement of proteins in plasma or serum derived from circulating blood. The dynamic range of protein concentration in these samples, and the dominance of very high abundance proteins such as albumin and immunoglobulins, make measurement of low concentration proteins impossible without some type of enrichment approach. This workshop will discuss the benefits of using Seppro® protein depletion columns in sample preparation for multiplexed LC-MS protein clinical assays.Read more >
Enabling Reliable Compound Identification using LC-MSn
Reliable compound identification with non-targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a major challenge. It involves matching against reference data. For fast and automated identification, tandem mass spectral libraries are queried with appropriate search tools. Reliability of search as well as time and efforts spent for data reviewing very much depend on the quality of the database involved. In this presentation, we will give an overview on our efforts towards the development of a reliable, robust, and transferable tandem mass spectral database, and how such a database can successfully be implemented in workflows for comprehensive drug analysis.
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.
DEVELOPMENT OF RAPID LC/MS/MS-BASED METHODS FOR CONFIRMATORY ANALYSIS OF OPIATES AND BENZODIAZEPINES
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
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.Read more >
Harnessing the Power of High-Resolution, Accurate Mass LC/MS Technology in Food Safety Laboratories
In this webinar, you will learn the advantages of high-resolution, accurate mass LC/MS technology and how it can be applied in the food safety laboratory. Instrumentation that offers high resolving power and ultimate mass accuracy provide unique advantages in screening and quantifying low levels of contaminants in complex food matrices. Software is the key to efficient processing of this data to obtain quantitative and qualitative results. Attend this webinar to learn what features to look for in a high-resolution solution for your lab:
-- What do you need to streamline your workflow and increase throughput?
-- How are results verified so you have a high level of confidence in your results?
-- How can you screen for unknowns?
Mass spectrometry-based protein assays impart increased specificity and more rapid development times versus traditional methods, such as ELISA. Coupled with immunoaffinity enrichment, LC-MS/MS is becoming a powerful tool for the quantitation of proteins in plasma. Such methods typically rely on synthetic stable isotope labeled (SIL) peptide internal standards to correct for instrumental variability. For more accurate protein quantitation by LC-MS/MS, experimental variations throughout the entire sample preparation workflow, including protein fractionation, immunoaffinity enrichment, and enzymatic digestion, must be accounted for. An ideal way of improving assay reproducibility is to add a full-length stable isotope labeled recombinant protein, that is equivalent to the native target protein, to the sample at the initial stage of the assay workflow. We have developed a set of stable-isotope-labeled monoclonal antibodies expressed in CHO cells as well as SIL versions of several clinically-relevant human proteins expressed in E. coli, such as IGF1, and in mammalian HEK293 cells, such as Thyroglobulin (manufactured as a Certified Reference Material). We will present data to demonstrate that the use of full-length SIL proteins and antibodies as internal standards allows for more accurate and rapid quantitation of biotherapeutic antibodies and clinically-relevant human protein biomarkers in plasma by LC-MS/MS.Read more >
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.
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.
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.Read more >
Zirconia-coated silica particles selectively remove phospholipids from plasma. These particles use Lewis acid-base interactions to strongly bind phospholipids. HybridSPE technology can be used to remove both phospholipids and proteins from plasma samples in a simple 3-step protocol, resulting in better LC-MS sensitivity and longer HPLC column life.Read more >