Microscale Solid Phase Extraction Research Articles

Transient isotachophoresis–Capillary zone electrophoresis–Mass spectrometry method with off-line microscale solid phase extraction pretreatment for quantitation of intact low molecular mass proteins in various biological fluids

Quantification of proteins is still predominantly done by the traditional bottom-up approach. Targeting of intact proteins in complex biological matrices is connected with multiple challenges during the sample pretreatment, separation, and detection step of the analytical workflow. In this work, we focused on the development of an on-line hyphenated capillary zone electrophoresis-mass spectrometry method employing off-line microscale solid-phase extraction based on hydrophilic lipophilic balance (HLB) sorbent as a sample pretreatment step for the analysis of low molecular mass intact proteins (<20 kDa) spiked in various biological fluids (human serum, plasma, urine, and saliva). A detailed optimization process involved the selection of a suitable capillary surface, background electrolyte (BGE), and comparison of two in-capillary preconcentration methods, namely transient isotachophoresis (tITP) and dynamic pH junction (DPJ), to enhance the sensitivity of the method. Optimum separation of the analytes was achieved using uncoated bare fused silica capillary employing 500 mM formic acid (pH 1.96) + 5 % (v/v) acetonitrile as BGE. tITP was utilized as an optimum preconcentration technique, achieving a 19- to 127-fold increase in the signal intensity when using 200 mM ammonium formate (adjusted to pH 4.00) as the leading electrolyte and BGE as the terminating electrolyte. Off-line microscale solid-phase extraction with various eluate treatment procedures was evaluated to ensure the compatibility of the sample pretreatment method with the selected in-capillary preconcentration, separation, and detection process. Achieved extraction recoveries of spiked proteins were in the range of 76–100 % for urine, 12–54 % for serum, 21–106 % for plasma, and 25–98 % for saliva when the eluate was evaporated and reconstituted in the solution of the leading electrolyte to achieve the tITP process. The optimum method was validated across different biological matrices, offering good linearity, accuracy, and precision, and making it suitable for proteomic studies (e.g., therapeutic drug monitoring, biomarker research) in different biological samples.

Analysis of lipopolysaccharides by coupling microscale solid-phase extraction with capillary electrophoresis-laser induced fluorescence

Lipopolysaccharides (LPS), also known as endotoxins, can be released into the environment when the bacteria’s cell wall breaks apart, which induces inflammatory responses in humans at amounts of 1–10 ng kg−1. This paper reports on the development and optimization of a novel analytical method for the analysis of LPS based on the coupled use of a laboratory–made microscale solid-phase extraction (μSPE) device for the purification and enrichment of LPS before fluorescent dye derivatization and lastly capillary electrophoresis with laser-induced fluorescence (CE-LIF) for separation and detection. In this work, the limits of detection and quantification of Klebsiella pneumoniae (KP)-LPS were found to reach 1.32 ng mL−1 and 4.39 ng mL−1, respectively with a linear dynamic range of 0.125–0.625 mg L−1 and a relative standard deviation of 0.9% in migration time. Scanning electron microscopy analysis clearly supports the successful entrapment of KP-LPS within the sorbent before elution. In contrast to other LPS extraction methods, the optimized μSPE-based method coupled with CE-LIF provides the following advantages: small sample amount for sensitive and selective detection in CE-LIF (μSPE preconcentration factor of 23 times), reduced volumes of organic solvents used (175–225 µL), high recovery rate of 98%, as well as portability for on-site extraction and quantification of analytes. The method was successfully applied in the analysis of three types of real water samples (i.e. deionized water, secondary effluent and raw sewage). Raw sewage was found to contain the highest KP-LPS amount of 12.5 ng mL−1 followed by secondary effluent of 7.3 ng mL−1.

Genotype and Phenotype of Caffeine Metabolism in Identical Twins

A wide range of individual variability to caffeine's effects have been reported in the literature, with factors including gene variants, gender, and personal habits modulating the response to caffeine. For example, the literature suggests that a single nucleotide polymorphism (SNP) in intron 1 of the CYP1A2 gene is associated with “fast” versus “slow” caffeine metabolism. The goal of our study was to genotype and phenotype identical twins to investigate environmental versus genetic influences on caffeine metabolism. For genotyping, we are using PCR‐RFLP to determine C/A at rs762551. For phenotyping, we are using a micro‐scale solid phase extraction of caffeine from saliva, determining the concentration of caffeine via GC‐MS, and using the loss of caffeine over time to monitor the activity of CYP1A2, the principal enzyme for caffeine metabolism. Initial results suggest a good correlation of caffeine half‐lives within identical twin pairs. However, it was not possible to predict this half‐life based on rs762551 genotype. Our findings indicate a strong influence of behavioral factors such as daily caffeine consumption on CYP1A2 activity. These findings could have significance for optimizing the ergogenic effects of caffeine dosing.Support or Funding InformationInstitutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM0103423This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Highly efficient DNA extraction and purification from olive oil on a washable and reusable miniaturized device

Sample preparation from complex matrixes with minute DNA content could highly benefit from the miniaturization of solid phase extraction (SPE) based devices due to an increased surface area-to-volume ratio. However, the adaptation of “bench-top” based protocols for DNA purification to miniaturized devices is not as straightforward as it might seem, and several issues need to be considered. A careful evaluation of DNA extraction and purification protocols needs to be performed, taking into account the complexity of such samples, and in order to facilitate the integration with the subsequent step, normally DNA amplification. In this work a washable and reusable miniaturized device for DNA purification based on microscale solid phase extraction (μSPE), containing a commercial disposable silica membrane as the solid phase for DNA capture, was developed. The DNA purification protocol was firstly optimized by testing a set of different conditions, including buffer composition in all three steps of analysis and incubation during the elution step, with the objective of increasing the DNA yield and facilitating the integration in a miniaturized setting. This protocol was then tested with olive oil samples, including a pretreatment step also developed and optimized in this work. DNA analysis of olive oil samples is of high interest for the detection of fraudulent adulteration with oil from other seeds and for cultivar identification. The results were compared with the commercial NucleoSpin® Food kit regarding efficiency and purity of the DNA extract, by estimating the DNA yield and evaluating the absorbance ratios A260/280 and A260/230. The miniaturized DNA purification device showed better performance than the commercial kit tested, making this method a very promising sample preparation approach for olive oil and other samples with minute DNA content.

A microscale solid-phase extraction poly(dimethylsiloxane) chip for enrichment and fluorescent detection of metal ions

A rapid and simple enrichment system was developed on microfluidic chip which was integrated with on-line complexing and fluorescence detection. Microparticles of ion-exchange resin were trapped into the microchannel by a fabricated weir-structure in the end of the microchannel to construct a micro-solid-phase extraction (μ-SPE) device. Some commonly existing metal ions in environment were served as models to evaluate the performance of the proposed microdevice, in combination with on-line derivatization with 8-hydroxyquinolin-5-sulfonic acid (HQS) and fluorescence detection. The concentration and pH value of HQS solution were optimized for metal-HQS fluorescent derivatization. The parameters, which affected the efficiency of the developed method, including composition and concentration of eluent, pH value and the flow rate of HQS solution and elution, were also investigated. Under the optimal conditions, Ca(2+), Mg(2+), Zn(2+) and Pb(2+) were successfully determined by the μ-SPE device on-chip. The experimental enrichment factors for Ca(2+), Mg(2+), Zn(2+) and Pb(2+) were up to 520, 565, 578 and 487 folds, respectively.

Quantification of endogenous brassinosteroids in plant by on-line two-dimensional microscale solid phase extraction-on column derivatization coupled with high performance liquid chromatography–tandem mass spectrometry

An on-line two-dimensional microscale solid phase extraction (2DμSPE)-on column derivatization (OCD)-high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) method was developed for quantification of brassinosteroids (BRs) in plant tissues. Five BRs with widest distribution in plant species and high bioactivity (24-epibrassinolide, 24-epicastasterone, 6-deoxo-24-epicastasterone, teasterone and typhastero) were selected as target analytes. 2DμSPE column packed sequentially with phenyl boronic acid silica sorbent (the first dimension) and C18 silica sorbent (the second dimension) was used to selectively extract and enrich BRs by 110–146 times. OCD was carried out on the second dimension of 2DμSPE column with m-aminophenylboronic acid (m-APBA) as a derivatization reagent, enhancing the sensitivity of MS/MS to BRs by 13–8437 times. It was also found that pre-trap of derivatization reagent on the C18 section of 2DμSPE column could increase reaction efficiency by 3–10 times. The whole process time of the on-line system was less than 30min. The detection limits of the method for five BRs were between 1.4 and 6.6pg with RSDs less than 10%. Endogeneous BRs in tomato leaves were analyzed by using this method. Owing to the high selectivity of this on-line 2DμSPE system, BRs in plant extracts could be quantified using matrix-free standard calibration method with relative recoveries in the range of 80–124%.

Micropreconcentration units based on carbon nanotubes (CNT)

Carbon nanotubes (CNT) have some highly desirable sorbent characteristics which make them attractive for a variety of analytical applications. High adsorption capacity and rapid desorbability make CNT excellent candidates for micro-scale devices for gas and liquid-phase analysis. In gas-phase analysis one can implement a micro-concentrator or a micro-sorbent trap, which have been used in a variety of on-line chromatography and sensing applications. Interesting liquid-phase microtrapping applications include micro-scale solid-phase extraction (μ-SPE) and solid-phase micro extraction (SPME). In addition, the ease of surface functionalization, self assembly by chemical vapor deposition, and the formation of diverse polymer composites may well make CNT the high-performance sorbent of the future.

Online Hyphenation of Multimodal Microsolid Phase Extraction Involving Renewable Molecularly Imprinted and Reversed-Phase Sorbents to Liquid Chromatography for Automatic Multiresidue Assays

Molecular imprinted polymers (MIP) have recently drawn much attention as highly selective solid-phase materials for handling and isolation of organic pollutants in complex matrices. Because of the impaired retention capacity for target species as compared with reversed-phase materials and irreversible sorption of interfering compounds by nonspecific interactions, the implementation of MIP-based solid-phase reactors as permanent components in automatic flow-systems has not received widespread acceptance as of yet. To tackle this limitation, a dynamic microscale solid phase extraction (microSPE) method capitalizing on the principle of programmable flow and bead injection analysis is herein proposed as a front end to liquid chromatography for multiresidue assays. It involves in-line renewable tandem-SPE microcolumns composed of molecularly imprinted polymers and copolymeric N-vinylpyrrolidone/divinylbenzene beads integrated within the flow network for multimodal extraction. Chlorotriazine herbicides (namely, atrazine, simazine, propazine) and principal degradation products thereof (namely, deisopropylatrazine and deethylatrazine) were selected as model analytes. The effect of several parameters, including the dimensions and chemical composition of the sorptive microcolumns, the sample loading flow rate, the type and volume of eluent, the interface with liquid chromatography (LC), and the disposable nature of the column on the analytical performance were investigated in detail. The assembled flow setup features appropriate removal of interfering organic species via solvent switch with toluene, the circumvention of analyte band-broadening in LC by in-line merging of the eluate with a water stream, and the transfer of the overall analyte-containing eluate into the LC. For 10-mL sample percolation, limits of detection (S/N = 3) of 0.02-0.04 ng mL(-1), limits of quantification (S/N = 10) of 0.07-0.12 ng mL(-1), absolute recovery percentages >79%, precision within 1.4-5.5%, and enrichment factors of 46-49 were obtained for the suite of assayed herbicides. The multimodal microSPE method with renewable beads was applied to the multiresidue determination of the target herbicides in crude soil extracts and untreated environmental waters at concentration levels below those endorsed by the current EU Water Framework Directives following appropriate sample preconcentration and/or cleanup.

Microscale solid phase extraction of glyphosate and aminomethylphosphonic acid in water and guava fruit extract using alumina-coated iron oxide nanoparticles followed by capillary electrophoresis and electrochemiluminescence detection

A microscale solid-phase extraction (SPE) method using alumina-coated iron oxide nanoparticles (Fe 3O 4@Al 2O 3 NPs) as the affinity adsorbent for glyphosate (GLY) and its major metabolite aminomethylphosphonic acid (AMPA) in aqueous solution is reported. One milligram of Fe 3O 4@Al 2O 3 NPs was employed to extract both analytes in 5 ml of aqueous solution. After 5 min extraction, magnetic NPs were isolated from sample solution by employing an external magnet. Followed by rinsing the NPs with 5 μl of 20 mM Na 4P 2O 7 solution for 5 min, the extract was directly analyzed using the derivatization-free CE-electrochemiluminescence (CE-ECL) method. With a sample-to-extract volume ratio of 1000, the enrichment factors for GLY and AMPA were 460 and 64, respectively. The limits of detection (LODs) were 0.3 and 30 ng ml −1 for GLY and AMPA in water, respectively. The developed method was applied to the analysis of GLY in guava fruit. The LOD of GLY in guava was 0.01 μg g −1. Total analysis time including sample pretreatment, SPE and CE-ECL was less than 1 h.

Online in situ analysis of selected semi-volatile organic compounds in water by automated microscale solid-phase extraction with large-volume injection/gas chromatography/mass spectrometry

A fully automated analytical method was developed for the online in situ analysis of selected semi-volatile organic compounds in water. The method used a large-volume injection/gas chromatography/mass spectrometry coupled with a fully automated microscale solid-phase extraction technique, which was based on x– y– z robotic techniques. Water samples were extracted by using a 96-well solid-phase extraction plate. For most analytes included in this study, the obtained linear calibrations ranged from 0.05 to 5.0 μg/L with correlation coefficients of 0.996–1.000, the method detection limits were less than 0.1 μg/L, and the relative recoveries were in the range of 70–120% with a relative standard deviation of less than 15% for fortified reagent water samples. The applications to chlorinated tap water, well water, and river water have been validated. The obtained results were similar to those resulting from fortified reagent water samples for all analytes except metribuzin, bromacil, aldrin, and methoxychlor. Matrix effects were observed for these analytes. In general, this fully automated analytical method was rugged, reliable, and easy to operate, and was capable of providing real-time data to water treatment and distribution systems as well as water reservation and protection systems. In addition, the method could reduce the analytical costs associated with sample collection, transportation, storage, and preparation.

A rapid sample preparation method for mass spectrometric characterization of N‐linked glycans

A rapid method for analysis of glycans of glycoproteins is presented. This method comprised deglycosylation, sample cleanup and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of glycans. The enzymatic deglycosylation of N-linked glycoproteins was enhanced in terms of speed and reproducibility using an enzyme-friendly surfactant. The released glycans were desalted using a micro-scale solid phase extraction (SPE) device packed with a hydrophilic interaction chromatography (HILIC) sorbent. Hydrophilic glycans were well retained by SPE, while salts and surfactants were removed from the sample. The glycans were eluted using 25-50 microL of solvent and analyzed directly without derivatization using MALDI-MS. MALDI quadrupole time-of-flight (Q-Tof) instrumentation was utilized for glycan profiling and structure characterization by tandem mass spectrometry (MS/MS). The presented method allows sensitive analysis of glycans benefiting from optimized deglycosylation reactions and efficient sample cleanup.

A sol–gel immobilization of nano and micron size sorbents in poly(dimethylsiloxane) (PDMS) microchannels for microscale solid phase extraction (SPE)

Sorbent particles consisting of nano and micro silica, and micron size octadecylsilica (ODS) were immobilized using sol–gel chemistry onto poly(dimethylsiloxane) (PDMS) microfluidic channels to serve as μ-chip solid phase extraction (SPE) devices. Extraction, preconcentration and purification of biological and chemical analytes were carried out using these. Micro and nano scale silica-immobilized μ-SPE were used for the extraction/purification of DNA from recombinant Escherichia coli crude lysate. The average DNA recovery was 77 ± 9% ( X ± R.S.D.) for the micron size silica particles and 70 ± 5% ( X ± R.S.D.) for the nano silica particles. The extracted DNA could be amplified by polymerase chain reaction (PCR) whereas the DNA from the crude lysate solution could not be. This was a testimony to the purification capability of the μ-SPE device. ODS immobilized μ-SPE were used to study the extraction efficiency (EE) and enhancement factor (EF) for three groups of organic compounds, aromatics, phenols and carboxylic acids. They showed poor recovery and low enrichment because the analytes sorbed into the PDMS and were not quantitatively extracted.

Microscale Solid-Phase Extraction and HPLC Separation of Purine Deoxyribonucleoside Monophosphate Adducts of Benzo[a]pyrene Diol Epoxide

Benzo[a]pyrene, a polycyclic aromatic hydrocarbon, is a potent mutagen and genotoxic compound. Activation of benzo[a]pyrene by oxidative metabolism in cells leads to the formation of highly reactive (±) anti benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxides, which bind covalently to DNA bases, in particular to the nucleophilic N2 atom of guanine and the N6 atom of adenine. Here we have investigated the recovery of eight different enantiomers of purine deoxyribonucleoside monophosphate benzo[a]pyrene diol epoxide adducts after a microscale solid-phase extraction (SPE). Microscale SPE was carried out using a pipette tip with a bed of C18 chromatograph packing material. We also tested the ability of microscale SPE to isolate adducts derived from the in vitro reaction of calf thymus DNA with (±) anti benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide. The recoveries of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide monophosphate adduct standards were good, varying between 65% and 92%, except for the trans BPDE-dGMP adduct derived from the (−) enantiomer of benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide, which presumably became decomposed to tetrols during the purification process. After using a neutral condition during the SPE treatment, the decomposition to tetrols of the trans BPDE-dGMP adduct could be avoided and the recovery increased to 79%.

Microscale solid-phase extraction system for explosives

A simple, semi-automated, microcolumn solid-phase extraction (SPE) system is optimized for the extraction, preconcentration and HPLC analysis of seven different explosives and explosive derivatives contaminating seawater, river water and well water samples. The microcolumns were constructed from 1/16 in. O.D. PTFE tubing (1 in.=2.54 cm) packed with 0.5–1.5 mg of SPE material, LiChrolut EN or Porapak R. The extraction system consisted of two syringe pumps and several solenoid valves. Optimal detection limits were realized when the sample water flow-rate was maximally increased within the limits of the pump, 5–10 ml/min (despite exceeding the breakthrough threshold of the SPE microcolumn), and when the eluate volume collected from the column was minimized, <5 μl (despite very low recovery percentages).