Solid-phase extraction (SPE) is a popular method for sample preparation in mass spectrometry. It purifies and concentrates samples prior to analysis. While SPE is effective, manual processing is time-consuming and error-prone. Automating SPE can increase throughput and reproducibility while reducing errors. Automated SPE allows laboratories to meet high-throughput demands for mass spectrometry without compromising data quality.
This webinar will provide pesticides residue analysts with valuable information on software method development and data processing for the analysis of pesticide residues in food for both LC–MS and GC–MS. Technical experts will review the latest in software advances to help with data interpretation and reporting.
This webinar will provide pesticides residue analysts with valuable information on software method development and data processing for the analysis of pesticide residues in food for both LC–MS and GC–MS. Technical experts will review the latest in software advances to help with data interpretation and reporting.
Use of automation to achieve high performance solid phase extractionGERSTEL
Despite 40 years of SPE using LC sorbents, LC principles have been ignored due to the lack of flow control in SPE devices. Variable flow results in variation in results. Internal standards are used to achieve meaningful results. Measuring absolute recovery against external standards to demonstrate absence of matrix effect (gold standard) isn’t done. With a new SPE device, this is changed. It uses a syringe to achieve both automation & accurate flow. With GERSTEL, SPE & LC/MS/MS is automated in a single parallel workflow. van Deemter curves are measured & SPE performed at flow achieving >99% absolute recovery. As a micro device, sample dry down isn’t needed for enrichment up to 200x. SPE is performed efficiently, economically, & with performance matching all LC knowledge of the last 50 years. Examples of laboratory testing using reverse phase & ion exchange SPE are provided.
1.Orthogonal” methods for analysis are needed in order to increase the probability that a primary assay has provided the separation (and recognition) of all peaks of interest.
2.A standardized procedure is described for the development of an “orthogonal” RP-LC separation , assuming that a primary RP-LC method for a given sample already exists.
3.An average change in resolution Rs > 3 for all adjacent peaks in the chromatogram seems likely (but not certain) to provide sufficient “orthogonality” to allow the recognition of any peaks in the “orthogonal” method that may have been overlapped and hidden in the primary method.
4.It has been demonstrated that HILIC provides different selectivity than RP-HPLC and is a useful tool for orthogonal method development.
5.Packed column SFC may provide higher separation efficiency and faster analyses with less consumption of organic solvent. SFC also offers chromatographic separation selectivity that is often similar to that of normal phase LC.
6.Results indicate that the CE method compares well with HPLC and can be used for the determination of carvedilol enantiomers in human serum. Although limits of quantitation are lower with HPLC, the CE assay offers the advantage of faster analysis times and low consumption of solvents
Separation techniques: column chromatography - Classroom activitiesXplore Health
In our day-to-day we use devices that are based on some techniques that are also used
in the lab. In this teaching unit you will learn how a separation technique such as column
chromatography is used for water filtration.
Key Learning Objectives:
- Identify the biggest time-consuming activities that occur in the Gas Chromatography-Mass Spectrometry (GC-MS) workflow
- Learn a modern approach to minimize the time an operator spends on the data review, reporting, and complex method development
Overview:
In the routine workflow of daily GC-MS operations, analysts spend the majority of their workday reviewing data and conducting maintenance activities. Today, many laboratories are also exploring the addition of MS/MS capabilities. Add the MS/MS dimension along with more complex method development to this workflow, and the analyst’s workload becomes even more challenging.
How can we mitigate this challenge? In this web seminar, we will demonstrate how the efficiency of data analysis can be improved through dynamic, interactive GC-MS data review and automated MS/MS method development. Additionally, we will illustrate some innovative ways to minimize downtime on the instrument for maintenance activities, whether planned or unplanned, to help alleviate this burden on the analyst. Common challenges and corresponding solutions will be presented throughout.
For more information: http://www.thermoscientific.com/isq
Metabolomics is often described as the study of “the complete set of low molecular weight intermediates, which are context dependent, varying according to the physiology, developmental or pathological state of the cell, tissue, organ or organism”. In fact, metabolomics is a new term for an old science in which classical biochemical concepts are investigated. New and unique to the current research that is being conducted is the combination with genomics information and full system biology. In this refocus we will discuss the challenges in today's metabolomics research and how to address them
Use of automation to achieve high performance solid phase extractionGERSTEL
Despite 40 years of SPE using LC sorbents, LC principles have been ignored due to the lack of flow control in SPE devices. Variable flow results in variation in results. Internal standards are used to achieve meaningful results. Measuring absolute recovery against external standards to demonstrate absence of matrix effect (gold standard) isn’t done. With a new SPE device, this is changed. It uses a syringe to achieve both automation & accurate flow. With GERSTEL, SPE & LC/MS/MS is automated in a single parallel workflow. van Deemter curves are measured & SPE performed at flow achieving >99% absolute recovery. As a micro device, sample dry down isn’t needed for enrichment up to 200x. SPE is performed efficiently, economically, & with performance matching all LC knowledge of the last 50 years. Examples of laboratory testing using reverse phase & ion exchange SPE are provided.
1.Orthogonal” methods for analysis are needed in order to increase the probability that a primary assay has provided the separation (and recognition) of all peaks of interest.
2.A standardized procedure is described for the development of an “orthogonal” RP-LC separation , assuming that a primary RP-LC method for a given sample already exists.
3.An average change in resolution Rs > 3 for all adjacent peaks in the chromatogram seems likely (but not certain) to provide sufficient “orthogonality” to allow the recognition of any peaks in the “orthogonal” method that may have been overlapped and hidden in the primary method.
4.It has been demonstrated that HILIC provides different selectivity than RP-HPLC and is a useful tool for orthogonal method development.
5.Packed column SFC may provide higher separation efficiency and faster analyses with less consumption of organic solvent. SFC also offers chromatographic separation selectivity that is often similar to that of normal phase LC.
6.Results indicate that the CE method compares well with HPLC and can be used for the determination of carvedilol enantiomers in human serum. Although limits of quantitation are lower with HPLC, the CE assay offers the advantage of faster analysis times and low consumption of solvents
Separation techniques: column chromatography - Classroom activitiesXplore Health
In our day-to-day we use devices that are based on some techniques that are also used
in the lab. In this teaching unit you will learn how a separation technique such as column
chromatography is used for water filtration.
Key Learning Objectives:
- Identify the biggest time-consuming activities that occur in the Gas Chromatography-Mass Spectrometry (GC-MS) workflow
- Learn a modern approach to minimize the time an operator spends on the data review, reporting, and complex method development
Overview:
In the routine workflow of daily GC-MS operations, analysts spend the majority of their workday reviewing data and conducting maintenance activities. Today, many laboratories are also exploring the addition of MS/MS capabilities. Add the MS/MS dimension along with more complex method development to this workflow, and the analyst’s workload becomes even more challenging.
How can we mitigate this challenge? In this web seminar, we will demonstrate how the efficiency of data analysis can be improved through dynamic, interactive GC-MS data review and automated MS/MS method development. Additionally, we will illustrate some innovative ways to minimize downtime on the instrument for maintenance activities, whether planned or unplanned, to help alleviate this burden on the analyst. Common challenges and corresponding solutions will be presented throughout.
For more information: http://www.thermoscientific.com/isq
Metabolomics is often described as the study of “the complete set of low molecular weight intermediates, which are context dependent, varying according to the physiology, developmental or pathological state of the cell, tissue, organ or organism”. In fact, metabolomics is a new term for an old science in which classical biochemical concepts are investigated. New and unique to the current research that is being conducted is the combination with genomics information and full system biology. In this refocus we will discuss the challenges in today's metabolomics research and how to address them
This slide explains about the type of Chromatographic Technique, mainly about HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) with its uses and medical application, Normal–phase chromatography, Reversed-phase chromatography (RPC), Size-exclusion chromatography, and Ion-exchange chromatography.
1. Enhancing Mass Spectrometry Sample Preparation with
Automated Solid-Phase Extraction
Wednesday, December 18, 2013
Print
Rohit Shroff
Tecan Schweiz AG
Application Specialist
Mass spectrometry (MS) instrumentation has advanced tremendously over the past
few years, placing considerable sample preparation demands on the laboratory.
Significantly, sample preparation methodologies have progressed at a much slower
rate, becoming the major bottleneck in high-throughput mass spectrometry protocols. This, along with the limitations
associated with manual processing, has hindered the uptake of MS innovation in the life science industry.
Solid-phase extraction explained
Laboratories working in the small-molecule field generally must be able to handle large numbers of samples from
several different biological matrices, including blood, urine, plasma and serum. They may use a range of different MS
sample-preparation techniques—solid-phase and liquid-liquid extraction, enzymatic hydrolysis, protein precipitation,
‘dilute and shoot’ and protein purification—with the final choice depending on the application, matrix, analyte(s) of
interest and sensitivity required.
Solid-phase extraction (SPE) is a popular means of purifying and concentrating samples prior to analysis by liquid
chromatography-mass spectrometry (LC-MS). SPE is used widely in the pharmaceutical industry, clinical labs and
academia for toxicology testing, food testing and environmental analysis. It can be performed in several different formats
(cartridge, disk and 96-well plate) on a variety of biological matrices, but in all cases the result is the same: extraction of
the compounds of interest by retention of the analyte(s) on the solid phase and removal of matrix interferences, or vice
versa.
Designing a successful experiment
A typical SPE experiment involves four steps: (1) conditioning the solid phase, (2) sample application, (3) washing the
solid phase to remove impurities and (4) elution of the analyte(s).
SPE method development begins with the selection of the most suitable solid phase for the analyte(s) of interest.
Options include reverse-phase, normal-phase, ion-exchange and mixed-mode SPE. With a range of materials available
in each of these categories, developing a successful extraction methodology is highly dependent on selecting the correct
phase, and the properties of the analyte(s) of interest must be carefully considered.
Reverse-phase SPE is ideal for isolation of nonpolar analytes from polar matrices, and normal-phase SPE generally is
used to extract polar analytes from non-polar matrices. For charged analytes, ion-exchange SPE is the method of
choice; this may be cation or anion exchange, depending on whether the analyte is positively or negatively charged.
Mixed-mode SPE typically is used when a combination of neutral, acidic and basic compounds needs to be isolated from
a single complex matrix for subsequent separation.
Having selected the most appropriate SPE phase for the analyte or analytes of interest, it is important to establish the
correct bed weight for optimal extraction. As a rule of thumb, the mass of the analytes to be extracted should not exceed
4% to 5% of the mass of packing in the SPE consumable to avoid overloading the phase. For example, a maximum of 5
mg of analyte can be loaded onto a 1 ml SPE cartridge containing 100 mg of solid phase.
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2. Sample pH is another crucial consideration when selecting the solid phase, playing a key role in efficient interaction
between the phase and the analyte. Silica-based SPE phases are generally only stable between pH 2.5 and 7.5, and
sample pH may need to be adjusted prior to loading onto these phases. Alternatively, if experiments must be conducted
at extreme pH values outside this range, the more stable polymer-based phases may be a better choice.
Depending on the matrix, some form of sample pretreatment may be necessary prior to SPE extraction. Urine samples,
for example, may require straightforward dilution and/or hydrolysis to ensure solvation of the analytes of interest. For
other biological matrices, such as plasma, serum and whole blood, a protein-precipitation step, either by simple pH
adjustment or addition of a precipitant, may be required to release protein-bound analytes. Water samples typically do
not need any pretreatment beyond simple filtration, if a lot of solid material is present. Pretreated samples are then
applied to the SPE device, where careful choice of wash solvents enables any interfering compounds to be removed
without loss of analyte from the solid phase. Finally, the analytes are eluted from the solid phase for LC-MS analysis by
changing the elution strength of the solvent or, in the case of ion-exchange SPE, the pH.
A carefully developed SPE protocol offers several benefits. It is ideal for analyte enrichment, as large sample volumes
can be applied to the solid phase and the retained analytes eluted in a small volume of solvent, increasing the analyte
concentration in the aliquot for analysis. SPE also allows matrix interferences that would affect chromatographic analysis
to be removed, eliminating many co-eluting compounds that would otherwise mask the compounds of interest and make
detection and quantitation difficult or even impossible. In some cases, undetectable interferences can cause ion
suppression during LC-MS analysis; an optimized mixed-mode SPE protocol provides a very clean extract, minimizing
this effect.
Drawbacks of manual SPE
Very often, SPE is performed by hand. But manual SPE has some major limitations. It is cumbersome, time consuming
and prone to errors, resulting in poor reproducibility and sample recoveries as well as reduced productivity.
As mass spectrometry and chromatography have accelerated in recent years, slow sample-preparation procedures have
become a major bottleneck for high-throughput laboratories. Automation of SPE protocols can help overcome this
hurdle, offering many advantages. A diverse range of analytes and laboratory protocols can be accommodated,
increasing versatility and analytical flexibility, and enhancing throughput considerably. Typically, one liquid-handling
system can prepare sufficient samples for up to 10 mass-spectrometry systems on a daily basis, allowing laboratories to
meet high-throughput demands without compromising data quality. Additionally, the adoption of built-in security features,
such as sample tracking, satisfies even the most stringent quality requirements. Consequently, a variety of automated
devices have emerged, from small workstations processing six samples in parallel through to large, high-throughput
liquid-handling platforms—such as Tecan’s Freedom EVO® workstations—that are capable of performing parallel
extractions in 96-well plate format, enabling SPE protocols to be semi- or fully automated.
Typically, laboratories are looking for maximum throughput with excellent precision, seeking to minimize the number of
false positive and negative results and repeat analyses. SPE offers a simple means of isolating and concentrating
analytes from biological matrices, generating a clean extract for LC-MS analysis, and automation is the key to enhanced
sample throughput. By harnessing the power of automation, laboratories can enjoy benefits including full sample
traceability, increased throughput, improved turnaround times and enhanced reproducibility, as well as the almost
complete elimination of manual errors. That means fewer repeat analyses and, with full walk-away processing assured,
analysts are free to focus on what they do best: data interpretation.
Rohit Shroff is an Application Specialist at Tecan Schweiz AG
Related Products from: Tecan
SPE (Solid Phase Extraction)
PosID™ Positive Identification System
Te-VacS™ Vacuum Separation Module
Freedom EVOware® Sample Tracking
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3. Freedom EVO® Series Liquid Handling Workstations
Air LiHa (Air Displacement Pipetting Arm)
Workstation Modules »
Automated Solid Phase
Extraction (SPE) Systems
»
Bar Code Readers/Sample
Tracking Systems »
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