Combined Sample Preparation Research Articles

Interfacing commercially available capillary electrophoresis to sample preparation and/or detection systems to solve analytical problems

This review presents the state-of-the-art of interfacing sample preparation and detector systems with commercially available capillary electrophoresis (CE) equipment. The analytical potential of combined sample preparation units into commercially available CE equipment under integrated or coupling approaches, allowing at-line, on-line, and in-line arrangements to automate sample preparation is discussed. Some examples of the analytical applications are reported when using different sample treatment procedures, such as cleanup and preconcentration, extraction/filtering, gas extraction/diffusion, extraction with membrane and hollow fiber, and dialysis and extraction with supported liquid membrane. CE interfaced detectors are also discussed by distinguishing between commercially available CE equipment and those not commercially available and focusing on the latter. Interfaced CE approaches clearly expand the field of application of the CE and connect to the real world in routine analytical laboratories, avoiding the drawbacks and limitations of electrophoretic separations.

Reliable quantitation of β-hydroxyethoxyacetic acid in human urine by an isotope-dilution GC–MS procedure

An analytical method for the determination of β-hydroxyethoxyacetic acid (HEAA), the main urinary metabolite of 1,4-dioxane was developed and validated. The presented method involves liquid-liquid extraction of HEAA from the urine samples, followed by silylation and subsequent analytical separation and detection using GC-MS. The method is characterized by its simple and fast sample preparation in combination with a robust chromatography. The use of isotope dilution analysis enables an efficient compensation of matrix related effects and analyte losses due to sample workup. The excellent reliability and reproducibility of the method is demonstrated by the good accuracy and precision data. Within-day precision and day-to-day precision ranged from 0.6 to 1.2% and 1.5 to 2.6%, respectively. The mean relative recovery of the method was found to be 98-101%. The LOD and LOQ of HEAA were determined to be 0.2mg/L and 0.6mg/L, respectively. In summary, the presented analytical method is well suited to be used for routine biomonitoring of occupational exposure to 1,4-dioxane.

Multivariate study of parameters in the determination of pesticide residues in apple by headspace solid phase microextraction coupled to gas chromatography–mass spectrometry using experimental factorial design

Solid-phase microextraction (SPME) is a solvent-less sample preparation method which combines sample preparation, isolation, concentration and enrichment into one step. In this study, multivariate strategy was used to determine the significance of the factors affecting the solid phase microextraction of pesticide residues (fenobucarb, diazinon, chlorothalonil and chlorpyrifos) using a randomised factorial design. The interactions and effects of temperature, time and salt addition on the efficiency of the extraction of the pesticide residues were evaluated using 23 factorial designs. The analytes were extracted with 100μm PDMS fibres according to the factorial design matrix and desorbed into a gas chromatography–mass spectrometry detector. The developed method was applied for the analysis of apple samples and the limits of detection were between 0.01 and 0.2μgkg−1, which were lower than the MRLs for apples. The relative standard deviations (RSD) were between 0.1% and 13.37% with average recovery of 80–105%. The linearity ranges from 0.5–50μgkg−1 with correlation coefficient greater than 0.99.

Evaluation of the FilmArray ® system for detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis

AimsTo evaluate the sensitivity and specificity of the BioFire Diagnostics FilmArray® system in combination with their Biothreat Panel for the detection of Bacillus anthracis (Ba), Francisella tularensis (Ft) and Yersinia pestis (Yp) DNA, and demonstrate the detection of Ba spores.Methods and ResultsDNA samples from Ba, Ft and Yp strains and near-neighbours, and live Ba spores were analysed using the FilmArray® Biothreat Panel, a multiplexed PCR-based assay for 17 pathogens and toxins. Sensitivity studies with DNA indicate that the limit of detection is 250 genome equivalents (GEs) per sample or lower. Furthermore, the identification of Ft, Yp or Bacillus species was made in 63 of 72 samples tested at 25 GE or less. With samples containing 25 CFU of Ba Sterne spores, at least one of the two possible Ba markers was identified in all samples tested. We observed no cross-reactivity with near-neighbour DNAs.ConclusionsOur results indicate that the FilmArray® Biothreat Panel is a sensitive and selective assay for detecting the genetic signatures of Ba, Ft and Yp.Significance and Impact of the StudyThe FilmArray® platform is a complete sample-to-answer system, combining sample preparation, PCR and data analysis. This system is particularly suited for biothreat testing where samples need to be analysed for multiple biothreats by operators with limited training.

Investigating the role of microbes in mineral weathering: Nanometre-scale characterisation of the cell–mineral interface using FIB and TEM

Focused ion beam (FIB) sample preparation in combination with subsequent transmission electron microscopy (TEM) analysis are powerful tools for nanometre-scale examination of the cell–mineral interface in bio-geological samples. In this study, we used FIB-TEM to investigate the interaction between a cyanobacterium (Hassallia byssoidea) and a common sheet silicate mineral (biotite) following a laboratory-based bioweathering, incubation experiment. We discuss the FIB preparation of cross-sections of the cell mineral interface for TEM investigation. We also establish an electron fluence threshold (at 200keV) in biotite for the transition from scanning (S)TEM electron beam induced contamination build up on the surface of biotite thin sections to mass loss, or hole-drilling within the sections. Working below this threshold fluence nanometre-scale structural and elemental information has been obtained from biotite directly underneath cyanobacterial cells incubated on the biotite for 3 months. No physical alteration of the biotite was detected by TEM imaging and diffraction with little or no elemental alteration detected by STEM–energy dispersive X-ray (EDX) elemental line-scanning or by energy filtered TEM (EF-TEM) jump ratio elemental mapping. As such we present evidence that the cyanobacterial strain of H. byssoidea did not cause any measurable alteration of biotite, within the resolution limits of the analysis techniques used, after 3 months of incubation on its surface.

High throughput sample preparation in combination with gas chromatography coupled to triple quadrupole tandem mass spectrometry (GC–MS/MS): A smart procedure for (ultra)trace analysis of brominated flame retardants in fish

In this study, gas chromatography (GC) coupled to triple quadrupole tandem mass spectrometry (MS/MS) operated in electron ionisation mode (EI) has been shown to be an effective tool for the (ultra)trace analysis of several representative brominated flame retardants (BFRs) including polybrominated diphenyl ethers (PBDEs), pentabromotoluene (PBT), pentabromoethylbenzene (PBEB), etc. in complex food and environmental matrices. Using this type of instrumentation, improved selectivity and sensitivity of the instrumental analysis was achieved. In addition to GC–MS/MS (EI), a GC–MS method employing QqQ as a single quadrupole in negative chemical ionisation (NCI) mode was also developed, as this technique might be preferred for those compounds where EI did not provide suitable (intensive enough) mass transitions (e.g., decabromodiphenyl ethane). Following the development of the GC–MS/MS method, a substantial simplification of the sample preparation method was achieved by employing an ethyl acetate QuEChERS-based extraction followed by silica minicolumn clean-up. Using this novel approach, six samples may be prepared in approx. one hour, thus significant time savings were achieved compared to routinely used methods. In addition, the method employs the reduced amounts of organic solvent and other chemicals. Under the optimised conditions, recoveries of all target analytes using both GC–MS/MS (EI) and GC–MS (NCI) were within the range of 70–119% and repeatabilities of the analytical procedure were ≤16% at all three spiking levels (0.1, 1 and 5μgkg–1). Regarding quantification limits (LOQs), as expected, a single quadruple operated in NCI provided significantly lower LOQs compared to EI. However, using the triple quadrupole mass analyser, comparable LOQs were achieved for both methods (0.005–1μgkg−1 and 0.005–0.1μgkg−1 for GC–MS/MS (EI) and GC–MS (NCI), respectively). Moreover, when highly selective mass transitions in GC–MS/MS (EI) were used for identification and quantification, a significant decrease of problematic interferences was observed compared to NCI where most of the compounds were quantified according to the less selective m/z 79 corresponding to a bromine atom.

A U–HPLC–ESI–MS/MS–Based Stable Isotope Dilution Method for the Detection and Quantitation of Methotrexate in Plasma

High-dose methotrexate (MTX) is used in the treatment of proliferative diseases such as acute lymphoblastic leukemia. Therapeutic drug monitoring of plasma MTX is important to monitor efficacy and adverse events. The authors aimed to develop a liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS)-based method to determine MTX in plasma for therapeutic drug monitoring and pharmacokinetic studies. Samples were analyzed using a Waters Acquity UPLC and Quattro Premier XE. A Waters Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used running an isocratic mobile phase of 21% methanol and 10 mM ammonium bicarbonate. The electrospray was operated in the positive ionization mode monitoring the following mass transitions: m/z 455.2 > 308.2 for MTX and m/z 458.2 > 311.2 for MTXd3. The analysis combined straightforward sample preparation, consisting of dilution and protein precipitation, with a 3-minute run time. The method was linear up to 50 μM (r > 0.99), and the coefficient of variation was <6% for intraday and <10% for interday precision. Average recovery was 99%. There were no significant matrix effects. The lower limit of quantitation, defined as the lowest concentration at which the coefficient of variation <20% and S/N > 1:10, was 5 nM. Method comparison with the Abbott TDx fluorescent polarization immunoassay (FPIA) showed excellent agreement, and a small but significant negative constant bias was detected (LC-MS/MS = 0.98 × FPIA - 7.3). [corrected] The authors developed a specific and sensitive stable isotope dilution LC-ESI-MS/MS method to monitor MTX concentrations in plasma within the clinically relevant range. The method can be easily applied in clinical laboratories because it combines straightforward sample pretreatment with LC-MS/MS.

Pressurized liquid extraction as a green approach in food and herbal plants extraction: A review

Pressurized liquid extraction is a “green” technology for the extraction of nutraceuticals from foods and herbal plants. This review discusses the extraction principles and the optimization of the extraction parameters that improves the extraction efficiency. The use of different solvent mixtures and other extraction additives to enhance the efficiency of the extraction are discussed. Dynamic mode of extraction in Pressurized liquid extraction, and the use of combined and hyphenated sample preparation and analytical techniques are presented. This work discusses how different studies used Pressurized liquid extraction to enrich phenolic compounds, lignans, carotenoids, oils and lipids, essential oils and other nutraceuticals from foods and herbal plants.

Combination of saponification with in-tube liquid–liquid extraction and dispersive liquid–liquid microextraction for determination of polybrominated diphenyl ethers in whole milk by gas chromatography-mass spectrometry

A simple, inexpensive and reliable analytical method was developed for the determination of polybrominated diphenyl ethers (PBDEs) in whole milk by gas chromatography-mass spectrometry (GC-MS). Matrix components such as proteins and fats in the whole milk were removed by saponification procedure and the combined PBDEs were released. Then, a simple one-step liquid–liquid extraction procedure in the centrifuge tube (in-tube LLE) was performed and PBDEs were extracted into petroleum ether. After centrifugation, dehydration and nitrogen drying of petroleum ether phase, the extract was dissolved in acetonitrile. The acetonitrile solution was directly used as dispersant for the subsequent dispersive liquid–liquid microextraction (DLLME). Under the optimal conditions, the enrichment factors (EFs) of PBDEs ranged from 270 to 307. The calibration was performed by use of matrix-matched calibration standards, the correlation coefficients were better than 0.99 and the limits of detection (LODs) ranged from 0.012 to 0.29 μg L−1. The recoveries for the whole milk samples analysis spiked at 1 μg L−1 were between 78% and 120% with relative standard deviations (RSDs) ranging from 1.7% to 11% (n = 5). In this study, a simple, valid and combined sample preparation method was applied in the analysis of PBDEs in whole milk sample and the satisfactory recoveries for PBDEs were obtained.

Tracing Cationic Nutrients from Xylem into Stem Tissue of French Bean by Stable Isotope Tracers and Cryo-Secondary Ion Mass Spectrometry

Fluxes of mineral nutrients in the xylem are strongly influenced by interactions with the surrounding stem tissues and are probably regulated by them. Toward a mechanistic understanding of these interactions, we applied stable isotope tracers of magnesium, potassium, and calcium continuously to the transpiration stream of cut bean (Phaseolus vulgaris) shoots to study their radial exchange at the cell and tissue level with stem tissues between pith and phloem. For isotope localization, we combined sample preparation with secondary ion mass spectrometry in a completely cryogenic workflow. After 20 min of application, tracers were readily detectable to various degrees in all tissues. The xylem parenchyma near the vessels exchanged freely with the vessels, its nutrient elements reaching a steady state of strong exchange with elements in the vessels within 20 min, mainly via apoplastic pathways. A slow exchange between vessels and cambium and phloem suggested that they are separated from the xylem, parenchyma, and pith, possibly by an apoplastic barrier to diffusion for nutrients (as for carbohydrates). There was little difference in these distributions when tracers were applied directly to intact xylem via a microcapillary, suggesting that xylem tension had little effect on radial exchange of these nutrients and that their movement was mainly diffusive.

Characterization of IgG N-Glycans Employing a Microfluidic Chip that Integrates Glycan Cleavage, Sample Purification, LC Separation, and MS Detection

A novel polymeric microfluidic device with an on-chip enzyme reactor has been developed for the characterization of recombinant glycoproteins. The enzyme reactor chip packed with PNGase F-modified solid support material was combined with a microfluidic glycan cleanup chip and a commercially available HPLC-chip to perform glycoprotein deglycosylation, protein removal, glycan capture, glycan LC separation, and nanoelectrospray into a time-of-flight mass spectrometry (TOF-MS) system. With this integrated chip, the combined sample preparation and sample analysis time was reduced from multiple hours to less than 10 min. A once tedious and time-consuming glycan analysis workflow is now integrated into an HPLC-chip device. Glycan profiling analysis has been achieved with as little as 100 ng of monoclonal antibody. Furthermore, a single chip was shown to retain activity and perform equivalently for over 250 replicate glycan profiles from a recombinant antibody.

The determination of organophosphonate nerve agent metabolites in human urine by hydrophilic interaction liquid chromatography tandem mass spectrometry

A sensitive, robust isotope dilution LC/MS/MS method is presented for the quantitative analysis of human urine for the alkyl methylphosphonic acid metabolites of five organophosphorus nerve agents (VX, rVX or VR, GB or Sarin, GD or Soman, and GF or Cyclosarin). The selective sample preparation method employs non-bonded silica solid-phase extraction and is partially automated. While working with a mobile phase composition that enhances the electrospray ionization process, the hydrophilic interaction chromatography method results in a 5-min injection-to-injection cycle time, excellent peak shapes and adequate retention ( k′ = 3.1). These factors lead to limits of detection for these metabolites as low as 30 pg/mL in a 1-mL sample of human urine. The quality control data (15 and 75 ng/mL) demonstrate accurate (−0.5 to +3.4%) and precise (coefficients of variation of 2.1–3.6%) quantitative results over the clinically relevant urine concentration range of 1–200 ng/mL for a validation set of 20 standard and quality control sets prepared by five analysts over 54 days. The selectivity of the method is demonstrated for a 100-individual reference range study, as well as the analysis of relevant biological samples. The combined sample preparation and analysis portions of this method have a throughput of 288 samples per day.

Analysis of sensory quality changes during storage of a modified atmosphere packaged meat product (pizza topping) by an electronic nose system

The objective of this study was to determine whether an electronic nose could be used for measuring and modelling sensory quality changes in a pizza topping product during storage. A method involving a minimum of sample preparation in combination with a short sampling cycle mimicking an on-line situation was developed. A multivariate data analysis strategy involving principal component analysis (PCA) and partial least squares regressions (PLSR) was applied to determine the relationships between the electronic nose data, sensory analysis data and storage time. The results showed that the electronic nose was capable of detecting quality changes related to odour in the early stage and again from half-way to the last stage of the storage time, whereas the trained sensory panel could detect quality changes during the whole storage time. Applicability of the electronic nose for modelling the sensory perceived quality of the pizza topping product showed to be very promising indicating strong relationships between the electronic nose data and the perceived changes in odours during storage.

A new method for the quantitation of propofol in human plasma: efficient solid-phase extraction and liquid chromatography/APCI-triple quadrupole mass spectrometry detection

Propofol (2,6-diisopropyl phenol) is widely used for the induction and maintenance of anesthesia. Analyses of its pharmacokinetics require simple and sensitive methods for quantitation of propofol in human plasma. Previously reported HPLC and GC methods are limited by cumbersome extraction steps. We describe a novel method that combines sample preparation by solid-phase extraction (SPE) with hydrophilic–lipophilic balance cartridges and analysis with a sensitive LC-APCI-triple quadrupole mass spectrometry (MS/MS) method for better quantitation. The absolute recovery of the analyte was greater than 96%. The limit of quantification for propofol in plasma at a signal-to-noise ratio of 10 was 5 ng/ml. The precision of the assay yielded coefficients of variation ranging from 2.9 to 5.3% and an accuracies of 99–105%. Our method advances the quantitative analysis of propofol in human plasma by combining simple, rapid and efficient SPE with specific and sensitive quantitation by HPLC with APCI-MS/MS detection.

Simultaneous determination of incretin hormones and their truncated forms from human plasma by immunoprecipitation and liquid chromatography–mass spectrometry

The incretins, glucose-dependent insulinotropic peptide (GIP 1–42) and glucagon-like peptide 1 (GLP-1 7–36), are involved in regulation of gastric emptying, glucose homeostasis, body fat regulation and the glucose-induced insulin secretion from the endocrine pancreas. After release in the circulation both peptides are rapidly degraded by the exopeptidase dipeptidyl peptidase IV (DP IV) to the inactive polypeptides GIP 3–42 and GLP-1 9–36. In vivo stabilization of the active incretins by orally available DP IV-inhibitors is now widely accepted as a new therapeutic approach in antidiabetic treatment. In order to demonstrate the pharmacodynamic effect of DP IV-inhibitors, it is necessary to measure the plasma levels of active and inactive forms of GIP and GLP-1. We previously described an immunoprecipitation method as sample preparation and concentration in combination with a LC–MS analysis for determination of active and inactive GIP. We could improve the efficiency and suitability of this method by reduction of the necessary sample volume to 1.0 ml and simultaneous measurement of GIP 1–42, GIP 3–42 and GLP-1 7–36, GLP-1 9–36, without loss of sensitivity. An LOQ of approximately 5 and 11 pmol/l was maintained for GIP and GLP-1, respectively.

Pre-PCR processing: strategies to generate PCR-compatible samples.

Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: influence of ionization type, sample preparation, and biofluid

The purpose of the present work was to evaluate the synergistic effect of ionization type, sample preparation technique, and bio-fluid on the presence of matrix effect in quantitative liquid chromatography (LC)-MS/MS analysis of illicit drugs by post-column infusion experiments with morphine (10-μg/mL solution). Three bio-fluids (urine, oral fluid, and plasma) were pretreated with four sample preparation procedures [direct injection, dilution, protein precipitation, solid-phase extraction (SPE)] and analyzed by both LC-electrospray ionization (ESI)-MS/MS and LC-atmospheric pressure chemical ionization (APCI)-MS/MS. Our results indicated that both ionization types showed matrix effect, but ESI was more susceptible than APCI. Sample preparation could reduce (clean up) or magnify (pre-concentrate) matrix effect. Residual matrix components were specific to each bio-fluid and interfered at different time points in the chromatogram. We evaluated matrix effect in an early stage of method development and combined optimal ionization type and sample preparation technique for each bio-fluid. Simple dilution of urine was sufficient to allow for the analysis of the analytes of interest by LC-APCI-MS/MS. Acetonitrile protein precipitation provided both sample clean up and concentration for oral fluid analysis, while SPE was necessary for extensive clean up of plasma prior to LC-APCI-MS/MS.

Peptidomics-based discovery of novel neuropeptides.

Modern proteomic methodologies have significantly improved the possibilities of large-scale identification of proteins. However, these methodologies are limited by their inability to reliably detect endogenously expressed peptides. We describe a novel approach of combining sample preparation, comprising focused microwave irradiation and mass spectrometric peptide profiling that has enabled us to simultaneously detect more than 550 endogenous neuropeptides in 1 mg of hypothalamic extracts. Automatic switching tandem mass spectrometry and amino acid sequence determination of the peptides showed that they consist of both novel and previously described neuropeptides. The methodology includes virtual visualization of the peptides as two- and three-dimensional image maps. In addition, several novel and known post-translational modifications of the neuropeptides were identified. The peptidomic approach proved to be a powerful method for investigating endogenous peptides and their post-translational modifications in complex tissues such as the brain. It is anticipated that this approach will complement proteomic methods in the future.

Analysis of procyanidins in chocolate by reversed-phase high-performance liquid chromatography with electrospray ionisation mass spectrometric and tandem mass spectrometric detection

The analysis of procyanidins in crude chocolate extracts by reversed-phase high-performance liquid chromatography (HPLC) with electrospray ionisation mass spectrometry (MS) is described in this report. Catechin monomers and procyanidin oligomers (dimers to hexamers) were identified according to the mass of the single charged pseudomolecular ion ([M–H] −). Identification was further confirmed by collision-induced dissociation MS–MS analysis, which in addition, permitted the identification of double charged pentameric, hexameric, and heptameric ions. This study demonstrates the capability of the combination of HPLC and modern ion trap mass analysers to significantly reduce sample preparation and analysis time in combination with high specificity and structural information for compound identification.

Immunochemical measurement of polychlorinated biphenyl compounds in transformer oils

Polychlorinated biphenyl (PCB) contaminated insulating oils represent a serious environmental problem to the power-transmission industry and there is much ongoing work directed at decommissioning such oils. The correct identification of contaminated oils is a key aspect of any decommissioning program. However, current analytical approaches are costly, complex, slow or are non-specific. This paper describes a combined sample preparation and PCB-specific immunoassay method for the measurement of these compounds in transformer oils that overcomes these shortcomings. A key aspect of the work centered on manipulating the sample such that it was compatible with the immunoassay process. Sample pretreatment using octadecyl carbon (C18) sorbents or washing with KOH/ethanol-sulfuric acid proved suitable with PCB recoveries of /spl les/100%. Aroclor (PCB mixture) contaminated transformer oils were assayed using both an in-house immunoassay and a commercially available test kit for PCB in soil and water. The in-house method proved reliable for the measurement of oils containing >35 /spl mu/g mL/sup -1/ Aroclor, but overestimated Aroclor levels in oils containing <10 /spl mu/g mL/sup -1/ analyte. The commercial kit tended to underestimate Aroclor concentrations yielding values between 23 and 63% of that expected. These differences were attributed to the different antibody preparations employed.