About 80% of the imprecision in analytical results has been attributed to the preanalytics stage of the assay. Sample preparation, including human manipulations, receives most of the blame. Improving data quality was a common need expressed at the Fourth Annual SLAS Conference and Exhibition in February and at Pittcon 2015 held in March. Notable advances in sample preparation in the life sciences, environmental and liquid handling arenas are described below.
Life sciences
BRTI Life Sciences
Growing live 3D tissue or tumors on a chip to test drug candidates for safety and efficacy naturally leads to questions about sampling. BRTI Life Sciences (Two Harbors, Minn.) offers a kit to extract secreted proteins and another to isolate secreted RNA. The latter, Cell-Mate3D, produces RNA samples suitable for quantitative PCR, mRNA sequencing and other RNA assays.
Shimadzu
Reproducibility and detection limits in matrix-assisted laser desorption ionization (MALDI) mass spectrometry for ’omics can be improved with uniform coating of the matrix on the target plate. The iMLayer matrix vapor deposition system from Shimadzu Scientific Instruments (Columbia, Md.) uses sublimation to uniformly cover the target plate with fine grains of the matrix. The target plate is placed in a vacuum chamber. A mask protects the plate during the start and end of the sublimation cycle. Matrix thinness is controlled via laser reflectance off a glass slide. The result is better resolution and matrix coating uniformity than can be achieved with spray methods. Compatible matrices include DHB, 9-AA and CHCA.
Tecan
Tecan’s (Männedorf, Switzerland and San Jose, Calif.) Freedom EVO NGS workstation automates sample preparation for next-generation sequencing (NGS). It operates on the common sample types for DNA and RNA and delivers them in a matrix suitable for the TruSeq Nano DNA, TruSeq Standard mRNA and Nextera Rapid Capture Exome (Illumina, San Diego, Calif.) and the ion AmpliSeq and Ion Torrent NGS platforms (Life Technologies, Grand Island, N.Y.). The protocols were developed in collaboration with the University of North Carolina at Chapel Hill.
Environmental
Horizon Technology
Horizon Technology (Salem, N.H.) offers two membrane-drying devices for the DryDisk and DryDisk Barrel line. The DryDisk-R is an inexpensive and convenient replacement for drying extracts with sodium sulfate.
Figure 1 – SPE-DEX 3100 Oil & Grease Extractor from Horizon Technology.The Horizon SPE-DEX 3100 Oil & Grease Extractor (Figure 1) features improved ergonomics and handling of complex samples. It is compatible with the Speed-Vap IV automated evaporation system and Solvent TrapOG solvent recovery system, providing an integrated workflow for sample preparation.
An accessory for the SPE-DEX 3100, the WaterTrap makes it possible to dry extracts in-line after the solid-phase extraction (SPE) disk and automates what was once a tedious manual step involving sodium sulfate.
The Solvent TrapSVOC solvent recovery system provides a recovery rate of 95% for dichloromethane, a hazardous extraction solvent that is costly to buy and dispose of. The system incorporates the DryVap inline drying and evaporation unit to complete the dry-evaporate trap workflow.
LCTech GmbH/Pickering Laboratories
Sample preparation protocols for the assay of polychlorinated dioxins (PCDs) and polychlorinated biphenyls (PCBs) are complex and often labor intensive. The DEXTech automated extractor is designed for the sample preparation of PCBs and PCDs using automated liquid chromatography. The instrument is marketed by LCTech GmbH (Dorfen, Germany) and is distributed in the U.S. by Pickering Laboratories (Mountain View, Calif.).
Depending on the analyte, either a column packed with multilayer silica beds separated by glass fiber filters or one packed with Florisil (U.S. Silica Co., Berkeley Springs, W. Va.) is used. The first column retains fats and the second retains the PCD congeners, dioxins and furans. For PCB congeners, two carbon columns in series clean up the effluent from the Florisil column to provide a clean fraction with PCBs. The extraction system uses a stepdown transformer to run on 24 or 36 V. Lower voltage reduces spark hazard and may make the system more compatible with at-site sampling.
Shimadzu
Supercritical fluids, particularly carbon dioxide, are well suited for rapid extraction and purification of less polar compounds. Until Shimadzu integrated supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) in the Nexera UC (unified chromatography) system, the process and scale of the interface were not engineered for compatibility.
Shimadzu’s interface delivers the extract directly to an SFC. This can involve an MS detector for data-rich analyte identification. In particular, the sample quantizes are designed to match the analyte quantity to the sample size of the analytics. Transfer is automated. Cold spots that produce condensation are engineered out.
SRI Instruments Europe
An automated gas sampler from SRI Instruments Europe GmbH (Bad Honnef, Germany and Torrance, Calif.) feeds sample containers to gas chromatographs. The Greenhouse Workstation XYZ-Autosampling System features a 210-position vial rack for 20-mL headspace vials; up to 346 samples can be analyzed per day.
Biomics
In SPE cartridges, frits are often used to keep the sorbent bed in place, but they add dead volume and have been implicated in nonspecific adsorption of hydrophobic polymers. Biomics (Nantong, P.R. China and Agoura Hills, Calif.) developed a sorbent bed in which the beads are bonded together to form a tablet, and thus are not loose. The tablet fits tightly into the bore of the cartridge so that sample cannot leak around the tablet. Since flow is uniform and the bed is thin, less solvent is required for washing and elution, which reduces evaporation time.
PromoChrom Technologies
PromoChrom Technologies (Surrey, B.C.) offers instruments with single- and multiple-sample processing channels, some with inline capability. A wide range of sorbents in bulk and packed columns support the systems. Both channels have gravity and pressure-based elution, but gravity is recommended for very-low-throughput situations. Pressure-assisted SPE provides higher throughput and greatly improved reproducibility.1
Supelco
Supelco (Bellefonte, Penn., a division of Sigma-Aldrich [St. Louis, Mo.]) designed the economical C18 micro-pipet micro-extractor for LC sample prep. The pipet has active tips coated with C18 sorbent. Tip arrays are selectable up to a 96-well plate format. The adhesive that holds the C18 phase in place is not retentive to potential analytes.
The lifetime of solid-phase microextraction (SPME) fibers can be extended up to three times by coating the fiber with a nonpolar polymer that prevents the sorbent coating from overloading. The coating reduces matrix buildup on the fiber and seals the distal end of the SPE to inhibit sample wicking between the fiber and the SPME phase. SPME tips are available in a 96-well format to enhance compatibility with liquid handlers.
Supelco’s Supelclean EZ-POP NP is a dual-layer SPE cartridge optimized for the analysis of nonpolar persistent organic pollutants (POPs) in edible oil matrices. The target application is the extraction of polynuclear aromatic hydrocarbons (PAHs) from edible oils. The top layer is LC-Florisil, which extracts interferences with polar functional groups. Below the polyethylene frit, the lower layer is a mixture of Discovery C18 and Z-Sep sorbents that bind and remove fatty matrix interferences through hydrophobic interactions and Lewis acid–base interactions.
Molecularly imprinted polymers (MIPs) are SPE sorbents containing highly cross-linked polymers designed to selectively extract specific analytes or analyte classes. The SupelMIP SPE-Aminoglycosides MIP employs a sorbent that is specifically selective to the class of antibiotics categorized as aminoglycosides. Aminoglycoside antibiotics are often given to farm animals to improve weight gain or treat infection. They are also found in cell culture fluids, honey and other food products. SupelMIP SPE-Aminoglycosides has been used to reliably quantify 10 different aminoglycoside antibiotics in porcine tissue, honey, cell culture and beef kidney matrices.
SupelMIP SPE-Bisphenol A MIP selectively extracts bisphenol A (BPA) from broth or milk-based matrices. The selectivity provides a lower limit of detection and reduced sample prep time and labor.
Analab S.A.R.L.
Sample preparation often involves pipetting with air-driven pipets. The plastic tips are a potential source of contamination. Washing the tips with acid can remove surface contaminants that may arise during storage. Analab S.A.R.L. (Hoenheim, France) offers the Cold P’Tips cleaner for trace and ultratrace analyses. The simple PTFE device processes a rack of 96 tips per batch.
Biomics
Hollow-fiber liquid-phase microextraction (HF-LPME) is a powerful technique for extracting and concentrating dilute analytes. The hollow fiber, which is sealed at one end and attached to a syringe needle at the other, is filled with the acceptor solvent. Loaded fiber is immersed in the analyte containing matrix solution. The analyte passes through the membrane and into the accepting solvent. The technique is fast and easily automated. Biomics is the first vendor to offer HF-LPME products, which are marketed under the Baulo brand.
Environmental Express
Up to 26 tubes can be processed in parallel with the SimpleDist Micro from Environmental Express (Charlestown, S.C.).The system is designed to be used with the company’s disposable micro distillation tubes.
Glygen
Using the tag line, “Lab-in-a-Tip,” the NuTip enables micro-SPE. The sorbent is embedded in the inner the wall of the plastic pipet tip without the use of adhesives. Samples as small as 0.1 μL can be extracted.
Other products from Glygen include the TopTip, a micro spin column that contains a small packed bed with no matrix or frit, and the disposable PrickTip, or Needle-in-a-Tip, which features a stainless steel needle fixed at the bottom of a micro plastic pipet tip. PrickTip pierces the septum in the vials or closing mat of multiple-well plates. The needle is held mechanically without the use of glue or other polymers. Disposable InjecTips are scaled for HPLC injection. The needle can be loaded with a sorbent as in the NuTip above.
Grenova
TipNovus from Grenova (Richmond, Va.) sanitizes and washes batches of pipet tips in a benchtop workstation. Tips can be reused several times, resulting in a 90% reduction in consumables costs. Protocols and wash solutions for blood, plasma, serum and red blood cells are available.
Hamilton Robotics
The easyPunch STARlet liquid handling system from Hamilton Robotics (Reno, Nev.) automates sample collection card punching and then processes the extraction. All common dried blood spot cards are supported.
Polymer Char
The properties of polypropylene can be altered by the addition of other molecules with rigid physical properties. Polymer Char (Valencia, Spain) offers the CRYSTEX for sample preparation and assay of the xylene solubles in polypropylene copolymers. A weighed sample is placed into a 150-mL vial; then the CRYSTEX QC calculates and dispenses the required volume of xylenes. The crystalline amorphous fractions are separated by temperature-controlled cycling in a temperature rising elution fractionation (TREF) column. Detection is with an infrared detector, which obviates the need for sample mass measurement.
Rainin Instrument
Some samples, such as fluticasone, are notoriously sticky, while others, like DNA and proteins, may be present in very low quantities and can be nonspecifically adsorbed on pipet tips. Rainin Instrument (Oakland, Calif.), a subsidiary of Mettler-Toledo, developed Rainin LR (low-retention) pipet tips made from a fluoropolymer to form what the company refers to as a “superhydrophobic” surface. Rainin LR tips show less variation in pipetting results with changes in viscosity, surface tension and density.
When the tips were used in a study of diabetic retinopathy (in which the amount of sample proteins is very low), 28 marker proteins were distilled into two panels that were useful in patient stratification.
Summary
Quality data depends on having a representative sample combined with similar analytics. Purpose-built sample preparation products result in improved data quality and convenience.
Reference
- www.americanlaboratory.com/914-Application-Notes/172423-Evaluation-of-an-Automated-Solid-Phase-Extraction-Method-Using-Positive-Pressure/
Robert L. Stevenson, Ph.D., is Editor Emeritus, American Laboratory/ Labcompare; e-mail: [email protected].