Automated 96-well SPE and LC-MS-MS for determination of protease inhibitors in plasma and cartilage tissues

Bioanalytical methods based on automated solid-phase extraction (SPE) and high-performance liquid chromatography with electrospray tandem mass spectrometry (LC-MS-MS) have been developed and utilized for the determination of MMP inhibitors in plasma and cartilage tissues. The SPE methods were automated using a 96-well extraction plate and a 96-channel programmable liquid-handling workstation. The LC-MS-MS methods were developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the positive ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized SPE methods, crude plasma or ground cartilage supernatant samples were loaded onto an SPE plate to remove proteins and other interfering components in the matrixes to render relatively clean extracts for LC-MS-MS analysis. Compared to the simple plasma protein precipitation method, the automated SPE method afforded significant time-saving in sample preparation and improved sensitivity in MS detection. The methods were validated and successfully applied to the analysis of protease inhibitors in plasma and cartilage tissues.     

固相萃取富集—高效液相色谱法分析水中多环芳烃(PAHs)

采用固相萃取与高效液相色谱联用技术,测定了水中的多环芳烃。实验中使用Supelco固相萃取过滤装置和Supelco C_(18)固相萃取小柱,100％的甲醇作为流动相。对于萤蒽(FLU),苯并(b)萤蒽(BbF),苯并(K)萤蒽(BkF),苯并(ghi)(BPer)及茚并(1,2,3-cd)芘(IP)的检测限分别为4.1,3.8,1.6,14.4,和3.8ng/L。     

LC-UV-solid-phase extraction-NMR-MS combined with a cryogenic flow probe and its application to the identification of compounds present in Greek oregano

Structure elucidation of natural products usually relies on a combination of NMR spectroscopy with mass spectrometry whereby NMR trails MS in terms of the minimum sample amount required. In the present study, the usefulness of on-line solid-phase extraction (SPE) in LC-NMR for peak storage after the LC separation prior to NMR analysis is demonstrated. The SPE unit allows the use of normal protonated solvents for the LC separation and fully deuterated solvents for flushing the trapped compounds to the NMR probe. Thus, solvent suppression is no longer necessary. Multiple trapping of the same analyte from repeated LC injections was utilized to solve the problem of low concentration and to obtain 2D heteronuclear NMR spectra. In addition, a combination of the SPE unit with a recently developed cryoflow NMR probe and an MS was evaluated. This on-line LC-UV-SPE-NMR-MS system was used for the automated analysis of a Greek oregano extract. Combining the data provided by the UV, MS, and NMR spectra, the flavonoids taxifolin, aromadendrin, eriodictyol, naringenin, and apigenin, the phenolic acid rosmarinic acid, and the monoterpene carvacrol were identified. This automated technique is very useful for natural product analysis, and the large sensitivity improvement leads to significantly reduced NMR acquisition times.     

High-efficiency on-line solid-phase extraction coupling to 15-150-microm-i.d. column liquid chromatography for proteomic analysis

The ability to manipulate and effectively utilize small proteomic samples is important for analyses using liquid chromatography (LC) in combination with mass spectrometry (MS) and becomes more challenging for very low flow rates due to extra column volume effects on separation quality. Here we report on the use of commercial switching valves (150-microm channels) for implementing the on-line coupling of capillary LC columns operated at 10,000 psi with relatively large solid-phase extraction (SPE) columns. With the use of optimized column connections, switching modes, and SPE column dimensions, high-efficiency on-line SPE-capillary and nanoscale LC separations were obtained demonstrating peak capacities of approximately 1000 for capillaries having inner diameters between 15 and 150 microm. The on-line coupled SPE columns increased the sample processing capacity by approximately 400-fold for sample solution volume and approximately 10-fold for sample mass. The proteomic applications of this on-line SPE-capillary LC system were evaluated for analysis of both soluble and membrane protein tryptic digests. Using an ion trap tandem MS it was typically feasible to identify 1100-1500 unique peptides in a 5-h analysis. Peptides extracted from the SPE column and then eluted from the LC column covered a hydrophilicity/hydrophobicity range that included an estimated approximately 98% of all tryptic peptides. The SPE-capillary LC implementation also facilitates automation and enables use of both disposable SPE columns and electrospray emitters, providing a robust basis for automated proteomic analyses.     

Sample filtration,concentration, and separation integrated on microfluidic devices

Microfabricated devices integrating sample filtration, solid-phase extraction, and chromatographic separation with solvent programming were demonstrated. Filtering of the sample was accomplished at the sample inlet with an array of seven channels each 1 microm deep and 18 microm wide. Sample concentration and separation were performed on channels 5 microm deep and 25 microm wide coated with a C18 phase, and elution was achieved under isocratic, step, or linear gradient conditions. For the solid-phase extraction, signal enhancement factors of 400 over a standard injection of 1.0 s were observed for a 320-s injection. Four polycyclic aromatic compounds were resolved by open channel electrochromatography in under 50 s. Chip operation was unaffected by the presence of the 5-microm silica particles at the filter entrance.     

monitoring activity-dependent peptide release from the CNS using single-bead solid-phase extraction and MALDI TOF MS detection

To investigate dynamic peptidergic cell-cell communication, single micrometer-sized solid-phase extraction (SPE) beads were used to collect peptides from specific locations of well-characterized neurosecretory structures and even individual neuronal processes for off-line MALDI MS analyses. Peptide binding parameters of single SPE beads, including limits of collection, detection, and saturation capacity, were tested with 14C-labeled cytochrome c as well as with mixtures of multiple neuropeptides (bradykinin, Aplysia acidic peptide 1-20, and insulin). MALDI MS detection of secreted peptides was demonstrated in two well-characterized neurosecretory structures, the rat pituitary gland and single cultured Aplysia bag cell neurons. With cultured cells, precise placement of SPE beads allowed peptide collection from distinct neurites with spatial localization on the order of 200 microm, and SPE beads could be replaced within time frames that allowed analyte collection before and after cell stimulation paradigms. Comparison between pre- and poststimulation peptide profiles in both model systems allowed a directed strategy to determine which compounds were released with neuronal activity. Single SPE bead MALDI MS offers a novel approach to investigate peptide signaling that allows the detection and discovery of unknown intercellular signals secreted from a large variety of biological tissues.     

Microchip-based purification of DNA from biological samples

A microchip solid-phase extraction method for purification of DNA from biological samples, such as blood, is demonstrated. Silica beads were packed into glass microchips and the beads immobilized with sol-gel to provide a stable and reproducible solid phase onto which DNA could be adsorbed. Optimization of the DNA loading conditions established a higher DNA recovery at pH 6.1 than 7.6. This lower pH also allowed for the flow rate to be increased, resulting in a decrease in extraction time from 25 min to less than 15 min. Using this procedure, template genomic DNA from human whole blood was purified on the microchip platform with the only sample preparation being mixing of the blood with load buffer prior to loading on the microchip device. Comparison between the microchip SPE (microchipSPE) procedure and a commercial microcentrifuge method showed comparable amounts of PCR-amplifiable DNA could be isolated from cultures of Salmonella typhimurium. The greatest potential of the microchipSPE device was illustrated by purifying DNA from spores from the vaccine strain of Bacillus anthracis, where eventual integration of SPE, PCR, and separation on a single microdevice could potentially enable complete detection of the infectious agent in less than 30 min.     

A device for automated direct sampling and quantitation from solid-phase sorbent extraction cards by electrospray tandem mass spectrometry

A new solid-phase extraction (SPE) device in the 96-well format (SPE Card) has been employed for automated off-line sample preparation of low-volume urine samples. On-line automated analyte elution via SPE and direct quantitation by micro ion spray mass spectrometry is reported. This sample preparation device has the format of a microtiter plate and is molded in a plastic frame which houses 96 separate sandwiched 3M Empore sorbents (0.5-mm-thickness, 8-microm particles) covered on both sides by a microfiber support material. Ninety-six discrete SPE zones, each 7 mm in diameter, are imbedded into the sheet in the conventional 9-mm pitch (spacing) of a 96-well microtiter plate. In this study one-quarter of an SPE Card (24 individual zones) was used merely as a convenience. After automated off-line interference elution of applied human urine from 24 samples, a section of SPE Card is mounted vertically on a computer-controlled X, Y, Z positioner in front of a micro ion spray direct sampling tube equipped with a beveled tip. The beveled tip of this needle robotically penetrates each SPE elution zone (sorbent disk) or stationary phase in a serial fashion. The eluted analytes are sequentially transferred directly to a microelectrosprayer to obtain tandem mass spectrometric (MS/MS) analysis. This strategy precludes any HPLC separation and the associated method development. The quantitative determination of Ritalin (methylphenidate) from fortified human urine samples is demonstrated. A trideuterated internal standard of methylphenidate was used to obtain ion current response ratios between the parent drug and the internal standard. Human control urine samples fortified from 6.6 to 3300 ng/mL (normal therapeutic levels have been determined in other studies to be between 50 and 100 ng/mL urine) were analyzed and a linear calibration curve was obtained with a correlation coefficient of 0.9999, where the precision of the quality control (QC) samples ranged from 9.6% at the 24 ng/mL QC level to 1.2% at the 3000 ng/mL QC level, and the accuracy for the four levels of QC samples ranged from 98.1% to 100.3%. The QC samples were prepared at four concentrations which included 24, 240, 1200, and 3000 ng/mL, respectively. The run time per sample in this work was 1.5 min not including the sample preparation time.     

High-efficiency, two-dimensional separations of protein digests on microfluidic devices

High-efficiency, two-dimensional separations of tryptic digests were achieved using glass microfluidic devices. Following micellar electrokinetic chromatography (MEKC) separations in a 19.6-cm-long serpentine channel, the peptides were rapidly sampled into a 1.3-cm-long second-dimension channel, where they were separated by capillary electrophoresis (CE). The turns in the serpentine channel were asymmetrically tapered to minimize geometrical contributions to band broadening and to provide ample channel length for high-efficiency chromatographic separations. Analysis of rhodamine B injections routinely produced plate numbers of 230000 and 40000 in the first (MEKC) and second (CE) dimensions, respectively, corresponding to plate heights of 0.9 and 0.3 microm. The electric field strengths were 200 V/cm for MEKC and 2400 V/cm for CE. In analysis times less than 15 min, two-dimensional separation of bovine serum albumin tryptic digest produced a peak capacity of 4200 (110 in the first dimension and 38 in the second dimension). The system was used to identify a peptide from a tryptic digest of ovalbumin using standard addition and to distinguish between tryptic digests of human and bovine hemoglobin.     

Quantitative separation of aliphatic and aromatic hydrocarbons using silver ion--silica solid-phase extraction

A solid-phase extraction (SPE) method employing silver nitrate impregnated silica has been developed and evaluated for the separation of defined aliphatic and aromatic hydrocarbons from crude oils. The versatility of the SPE method is demonstrated using a light crude oil from the North Sea and a heavy crude oil from Orcutt field (Monterey, California, U.S.A.). The coefficients of variation for a number of geochemical parameters measured on both aliphatic and aromatic hydrocarbons were excellent. The separation efficiency of SPE is demonstrated using quantification of monoaromatic steroid hydrocarbons which are notoriously difficult to efficiently sequester into the aromatic hydrocarbon fraction using traditional liquid chromatographic procedure. The selectivity and efficiency of the SPE technique is comparable with that of silica gel TLC. However, losses of volatile compounds such as naphthalene are limited during SPE since the sample remains in solvent. We conclude that solid-phase extraction affords rapid sample turnover suitable for processing large sample numbers with high reproducibility.     

Spherical clay conglomerates: a novel stationary phase for solid-phase extraction and "reversed-phase" liquid chromatography

A new solid phase is presented to be used for the solid-phase extraction (SPE) of organic compounds from aqueous solutions and as a stationary phase for the separation of organic compounds in "reversed-phase" HPLC. The material consists of spherical clay conglomerates (SCCs) in the size ranges of 2-5, 5-10, and 10-20 μm. SCCs are especially well suited for the extraction and separation of aromatic compounds with electron-withdrawing substituents, because of the formation of specific electron donor-acceptor (EDA) complexes of such compounds with natural clay minerals. A series of nitroaromatic compounds (NACs), e.g., nitrophenols, and nitrotoluenes, served as probe substances for the characterization of the SPE with SCCs online coupled to a C18-HPLC-DAD system. Breakthrough volumes were > 1 L and method detection limits (MDLs) < 100 ng/L for compounds with moderate to high affinity towards clay minerals. The performance of the material is hardly affected by matrix effects and because of its excellent physical properties, i.e., regenerability and pressure-resistance, it meets the requirements for fully automated routine trace analysis of several primary pollutants, such as 6-methyl-2,4-dinitrophenol (DNOC) or 2,4,6-trinitrotoluene (TNT), in various natural waters. Offline SPE with SCCs was superior or equivalent to commercial SPE products for analysis of such compounds. Finally, SCCs are shown to be well suited as a stationary phase in reversed-phase HPLC. This opens a wide range of applications, e.g., as an easy and fast separation technique that is orthogonal to C18 reversed-phase HPLC.     

Thermoplastic microfluidic device for on-chip purification of nucleic acids for disposable diagnostics

A polymeric microfluidic device for solid-phase extraction (SPE)-based isolation of nucleic acids is demonstrated. The plastic chip can function as a disposable sample preparation system for different biological and diagnostic applications. The chip was fabricated in a cyclic polyolefin by hot-embossing with a master mold. The solid phase consisted of a porous monolithic polymer column impregnated with silica particles. The extraction was achieved due to the binding of nucleic acids to the silica particles in the monolith. The solid phase was formed within the channels of the device by in situ photoinitiated polymerization of a mixture of methacrylate and dimethacrylate monomers, UV-sensitive free-radical initiator, and porogenic solvents. The channel surfaces were pretreated via photografting to covalently attach the monolith to the channel walls. The solid phase prepared by this method allowed for successful extraction and elution of nucleic acids in the polymeric microchip.     

Application of porous membrane-protected micro-solid-phase extraction combined with HPLC for the analysis of acidic drugs in wastewater

This report describes the use of a porous membrane-protected micro-solid-phase extraction (micro-SPE) procedure to extract acidic drugs from wastewater that are then determined by high-performance liquid chromatography with ultraviolet detection. The micro-SPE device consists of C18 sorbent held within a membrane envelope made of polypropylene. Ketoprofen and ibuprofen were selected as model compounds, and extraction parameters were optimized. Correlation coefficients of 0.9980 and 0.9953 were obtained for ketoprofen and ibuprofen, respectively, across a concentration range of 1-250 microg/L. Relative extraction recoveries were between 94 and 112%. The relative standard deviation of the analytical method ranged between 2 and 10%, respectively. The method detection limits for these target analytes in wastewater ranged from 0.03 to 0.08 microg/L. When compared to conventional solid-phase extraction (SPE), this new method showed better detection limits with good reproducibility. The results shows that this micro-SPE technique is a feasible alternative to multistep SPE for the extraction of analytes in complex samples.     

Capillary electrophoresis with in-capillary solid-phase extraction sample cleanup

An in-capillary, solid-phase extraction (SPE)-capillary electrophoresis (CE) method, with not only high preconcentration factor but also no adsorption on the inner capillary wall of absorbing species in real complex samples, has been developed with a hole-opened capillary. The SPE sorbents approximately 3 mm in length was packed in the inlet end of the capillary. A hole approximately 30 microm in diameter was opened after the sorbents on the capillary. Sample solutions were loaded from the inlet end, and the sample wastes flowed out from the hole. After a certain time of the sample loading, a 1.5-mm-long methanol plug was introduced from the inlet end and forced to pass by the sorbents and the hole. Then, a separation voltage was applied between the hole and the outlet end of the capillary to carry out normal CE. When the sample loading time was 120 min, CE peak heights of the 2,4-dichlorophenol and 2,4,5-trichlorophenol were proportional to their concentration in a range of 0.08-5 ng/mL, and their detection limits were 25 and 17 pg/mL, respectively. A 16,000-fold sensitivity enhancement was obtained for CE of the chlorophenols with only a little decrease in CE separation efficiency. It was also demonstrated with the mixture of the chlorophenols and a surfactant cetyltrimethylammonium bromide that the present method could eliminate the adsorption problem of absorbing species on the inner wall during sample loading. Furthermore, the SPE-CE was directly applied to determination of chlorophenols on the level of 0.02 ppb in downstream water of a river, and the results agreed well with those obtained with off-line SPE-HPLC experiments.     

A low-makeup beveled tip capillary electrophoresis /electrospray ionization mass spectrometry interface for micellar electrokinetic chromatography and nonvolatile buffer capillary electrophoresis

A robust interface has been developed for interfacing micellar electrokinetic chromatography (MEKC) and nonvolatile buffer capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS). The interface consists of two parallel capillaries for separation (50 microm i.d. x 155 microm o.d.) and makeup (50 microm i.d. x 155 microm o.d.) housed within a larger capillary (530 microm i.d. x 690 microm o.d.). The capillaries terminate in a single tapered tip having a beveled edge. The use of a tapered beveled edge results in a greater tip orifice diameter (75 microm) than in a previous design from our laboratory (25 microm) that used a flat tip. While maintaining a similar optimum flow rate and consequently similar sample dilution, a 75-microm beveled emitter is more rugged than a 25-microm flat tip. Furthermore, the incorporation of a sheath liquid capillary allows the compositions of the final spray solution to be controlled. The application of this novel CE/ESI-MS interface was demonstrated for MEKC using mixtures of triazines (positive ion mode) and phenols (negative ion mode). The ability to perform CE/ESI-MS using a nonvolatile buffer was demonstrated by the analysis of gangliosides with a buffer consisting of 40 mM borate and 20 mM alpha-cyclodextrin.     

A simple, valveless microfluidic sample preparation device for extraction and amplification of DNA from nanoliter-volume samples

A glass microdevice has been constructed for the on-line integration of solid-phase extraction (SPE) of DNA and polymerase chain reaction (PCR) on a single chip. The chromatography required for SPE in the microfluidic sample preparation device (muSPD) was carried out in a silica bead/sol-gel SPE bed, where the purified DNA was eluted directly into a downstream chamber where conventional thermocycling allowed for PCR amplification of specific DNA target sequences. Through rapid, simple passivation of the PCR chamber with a silanizing reagent, reproducible DNA extraction and amplification was demonstrated from complex biological matrixes in a manner amenable to any research laboratory, using only a syringe pump and a conventional thermocycler. The muSPD allowed for SPE concentration of DNA from 600 nL of blood coupled to subsequent on-chip amplification that yielded a detectable amplicon; this simple device can be applied to a variety of routine genetic analyses without the need for sophisticated instrumentation. In addition, the applicability of these developments to nonconventional thermocycling was demonstrated through the use of noncontact, IR-mediated heating. This was exemplified with the isolation of DNA from an anthrax spore-spiked nasal swab and the subsequent on-chip amplification of target DNA sequences in a total processing time of only 25 min.     

Chlorinated phenol analysis using off-line solid-phase extraction and capillary electrophoresis coupled with amperometric detection and a boron-doped diamond microelectrode

The analysis of chlorinated phenols (2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, pentachlorophenol) in river water was accomplished using off-line solid-phase extraction (SPE) and capillary electrophoresis coupled with electrochemical detection. A key to the sensitive, reproducible, and stable detection of these pollutants was the use of a boron-doped diamond microelectrode in the amperometric detection mode. An off-line SPE procedure was utilized to extract and preconcentrate the pollutants prior to separation and detection, with ENVI-Chrom P, a highly cross-linked styrene-divinylbenzene copolymer, being employed as the sorbent. Pollutant recoveries in the 95-100% range with relative standard deviations of 1-4% were achieved. The diamond microelectrode provided a low and stable background current with low peak-to-peak noise. The oxidative detection of the pollutants was accomplished at +1.05 V vs Ag/AgCl without the need for electrode pretreatment. The method was evaluated in terms of the linear dynamic range, sensitivity, limit of quantitation, response precision, and response stability. A reproducible electrode response was observed during multiple injections of the chlorinated phenol solutions with a relative standard deviation of < or =5.4%. Good electrode response stability was observed over many days of continuous use with no significant electrode deactivation or fouling. The separation efficiencies for all six pollutants were greater than 170,000 plates/m. The minimum concentration detectable for all six ranged from 0.02 to 0.2 ppb (S/N > or = 3) using a 250:1 preconcentration factor.     

Two-dimensional electrophoretic separation of proteins using poly(methyl methacrylate) microchips

The work presented herein describes highly efficient, two-dimensional (2D) electrophoretic separations of proteins in a PMMA-based microchip. Sodium dodecyl sulfate microcapillary gel electrophoresis (SDS micro-CGE) and micellar electrokinetic chromatography (MEKC) were used as the separation modes for the first and second dimension of the electrophoresis, respectively. The microchip was prepared by hot embossing into PMMA from a brass mold master fabricated via high-precision micromilling. The microchip incorporated a 30-mm SDS micro-CGE and a 10-mm MEKC dimension length. Electrokinetic injection and separation were used with field strengths of up to 400 V/cm. Alexa Fluor 633 conjugated proteins, ranging in size from 38 to 110 kDa, were detected using laser-induced fluorescence with excitation/emission at 633/652 nm. Average plate numbers (N) of 4.8 x 10(4) and 1.2 x 10(4) were obtained in the SDS micro-CGE and MEKC separation dimensions, respectively, for the investigated proteins corresponding to plate heights (H) of 0.62 and 0.87 microm. Effluents from the first dimension (SDS micro-CGE) were repetitively transferred into the second dimension every 0.5 s of run time in the first dimension with the electrophoresis run time in the MEKC dimension being 10 s. The 2D separation was performed on the investigated proteins in approximately 12 min and provided a peak capacity of approximately 1000.     

Integrated label-free protein detection and separation in real time using confined surface plasmon resonance imaging

We demonstrate a label-free protein detection and separation technology for real-time monitoring of proteins in micro/nanofluidic channels, confined surface plasmon resonance imaging (confined-SPRi). This was achieved by fabricating ultrathin fluidic channels (500 nm high, 500 microm wide) directly on top of a specialized SPRi sensor surface. In this way, SPRi is uniquely used to detect proteins deep into the fluidic channel while maintaining high lateral accuracy of separated products. The channel fluid and proteins were driven electrokinetically under an external electric field. For this to occur, the metallic SPR sensor (46 nm of Au on 2 nm of Cr) was segmented into an array of squares (each 200 microm x 200 microm in size and spaced 8 microm apart) and coated with 30 nm of CYTOP polymer. In this work, we track label-free protein separation in real time through a simple cross-junction fluidic device with an 8-mm separation channel length under 30 V/cm electric field strength.     

Multiresidue herbicide analysis in soil: subcritical water extraction with an on-line sorbent trap

We evaluated the feasibility of extracting selectively and rapidly herbicide residues in soils by hot water and collecting analytes with a Carbograph 4 solid-phase extraction (SPE) cartridge set on-line with the extraction cell. Phenoxy acid herbicides and those nonacidic and acidic herbicides which are often used in combination with phenoxy acids were selected for this study. Five different soil samples were fortified with target compounds at levels of 100 and 10 ng/g (30 ng/g of clopyralid and picloram) by following a procedure able to mimic weathered soils. Herbicides were extracted with water at 90 °C and collected on-line by the SPE cartridge. After the cartridge was disconnected from the extraction apparatus, analytes were recovered by stepwise elution to separate nonacidic herbicides from acidic ones. The two final extracts were analyzed by liquid chromatography/mass spectrometry with an electrospray ion source. At the lowest spike level considered, analyte recoveries ranged between 81 and 93%, except those for 2,4-DB and MCPB, which were 63%. For 16 herbicides out of 18, the ANOVA test showed recoveries were not dependent on the type of soil. The method detection limit was in the 1.7-10 ng/g range. For the analytes considered, method comparison showed this extraction method was overall more efficient than Soxhlet and sonication extraction techniques.