Determination of theophylline in serum by molecularly imprinted solid-phase extraction with pulsed elution

The technique of molecular imprinting is used to produce an extensively cross-linked poly(methacrylic acid-co-ethylene dimethacrylate) material that contains theophylline as a print molecule. After Soxhlet extraction of the theophylline, binding sites are formed in the polymer with complementary size, shape, and positioning of chemical functionalities. The molecularly imprinted polymer's (MIP) high theophylline selectivity, chemical stability, and physically robust nature make it an ideal stationary-phase material in columns for HPLC separation of theophylline from other structurally related drug compounds. Mobile-phase tests confirm that a retention mechanism typical of normal-phase chromatography governs the separation, and selectivity of the MIP column can be controlled by a combination of the mobile phase and the sample solvent. Under optimal conditions, the MIP column functions like a solid-phase sorbent for theophylline extraction. Rapid elution of the bound theophylline can be accomplished in a pulsed format through injection of 20 μL of a solvent that has the proper polarity and protic nature to disrupt the electrostatic interactions and hydrogen bonding between theophylline and binding sites. A concentration detection limit of 120 ng/mL is obtained using direct UV absorption detection at 270 nm, which corresponds to a mass detection limit of 2.4 ng. This new technique, molecularly imprinted solid-phase extraction with pulsed elution (MISPE-PE), permits on-line preconcentration of theophylline from a large volume of dilute sample solution. Using a sample volume of 300 μL, a 40 ng/mL standard solution produces a detectable peak signal. Application of MISPE-PE in serum analysis further demonstrates the high capability of the MIP column to selectively isolate theophylline from other matrix components for fast, accurate determination.     

Multidimensional high-throughput screening of displacers

A multidimensional, batch high-throughput screening (MD-HTS) protocol was developed to investigate the effects of various parameters on the selectivity of ion-exchange protein displacement systems. A variety of molecules were screened, and the results were employed to provided insights into the influence of displacer chemistry and concentration, resin chemistry, and mobile-phase salt counterion on the efficacy and selectivity of these nonlinear chromatographic systems. These results open up the possibility of tailoring the selectivity of displacement separations by choosing appropriate combinations of operating conditions using the MD-HTS technique. The screens were also employed for the identification of displacers and conditions for the separation of a challenging protein mixture by selective displacement chromatography. Column displacements were carried out with potential lead compounds identified from the MD-HTS screens, and the results confirmed that selective displacement could indeed be achieved for this model mixture. Furthermore, the results indicated that this approach is particularly useful when the order of elution is not changed, but the inherent selectivity is increased in the presence of the displacer. The results presented in this paper demonstrate the utility of the MD-HTS technique for rapid method development in protein ion-exchange displacement chromatography.     

Fundamentals of capillary electrochromatography: migration behavior of ionized sample components

The mechanism of separating charged species by capillary electrochromatography (CEC) was modeled with the conditions of ideal/linear chromatography by using a simple random walk. The most novel aspect of the work rests with the assumption that in sufficiently high electric field ionized sample components can also migrate in the adsorbed state on the ionized surface of the stationary phase. This feature of CEC leads to the introduction of three dimensionless parameters: alpha, reduced mobility of a sample component with the electrosmotic mobility as the reference; beta, the CEC retention factor; and gamma, the ratio of the electrophoretic migration velocity and the velocity of surface electrodiffusion. Since the interplay of retentive and electrophoretic forces determines the overall migration velocity, the separation mechanism in CEC is governed by the relative importance of the above parameters. The model predicts conditions under which the features of the CEC system engender migration behavior that manifests itself in a relatively narrow elution window and in a gradient like elution pattern in the separation of peptides and proteins by using pro forma isocratic CEC. It is believed that such elution patterns, which resemble those obtained by the use of external gradient of the eluent, are brought about by the formation of an internal gradient in the CEC system that gave rise to concomitant peak compression. The peculiarities of CEC are discussed in the three operational modalities of the technique: co-current, countercurrent, and co-counter CEC. The results suggest that CEC, which is often called "liquid chromatography on electrophoretic platform" is an analytical tool with great potential in the separation of peptides and proteins.     

Behavior of microconcentrations of radionuclides in displacement complexing chromatographic systems

The behavior of microconcentrations of radionuclides (with Pm as example) in REE separation by displacement complexing chromatography was studied. The Pm elution curve is bell-shaped but obeys the laws of displacement chromatography. Addition of citric acid salts to the DTPA-based eluent makes the REE separation more efficient.     

Fractionation of carbon-based nanomaterials by anion-exchange HPLC

A sample containing carbon nanoparticles was obtained starting with the soot generated during combustion of inexpensive paraffin oil in a flame. The complexity of the sample, however, required fractionation to isolate its components. Anion-exchange high-performance liquid chromatography (AE-HPLC) was used for the analysis and collection of soot-derived carbon nanoparticles. The fractionated species were monitored by ultraviolet (UV) absorption and laser-induced photoluminescence detection, providing the chromatographic UV absorption and emission profiles of the separated sample. Chromatographic fractionation allowed for bulk measurements of electronic properties for individual fractions and further analysis via transmission electron microscopy (TEM). TEM of fractionated species showed a predominant size of about 4-5 nm diameter particulates. A general trend between photoluminescence and elution time was observed; the later eluting species in the chromatogram exhibited photoluminescence at longer wavelengths than the early eluting species. The AE-HPLC approach can have an immediate impact on the analysis and fractionation of various other nanomaterials, demonstrated here by analyzing samples containing graphitic oxide nanoparticles.     

Quantitative and wide-ranging profiling of phospholipids in human plasma by two-dimensional liquid chromatography/mass spectrometry

Normal-phase or reverse-phase liquid chromatography has been used in phospholipidomics for lipid separation prior to mass spectrometry analysis. However, separation using a single separation mode is often inadequate, as high-abundance phospholipids can mask large numbers of low-abundance lipids of interest. In order to detect and quantify low-abundance phospholipids, we present a novel two-dimensional (2D) approach for sensitive and quantitative global analysis of phospholipids. The methodology monitors individual glycerolipids and phospholipids through the use of a new quantitative normal-phase, solid-phase extraction procedure, followed by molecular characterization and relative quantification using an ion-trap Orbitrap equipped with a reverse-phase liquid chromatograph, with data processing by MS++ software. The CV (%) of the peak area of each lipid standard was less than 15% with this extraction method. When the method was applied to a liver sample, we could detect more phosphatidylserine (PS) compared to the previous method. Finally, our developed method was applied to Alzheimer's disease (AD) plasma samples. Several hundred peaks were detected from a 60 μL plasma sample. A partial-least-squares discriminant analysis (PLS-DA) plot using peak area ratio gave a unique group of PLS scores which could distinguish plasma samples of Alzheimer's disease (AD) patients from those of age-matched healthy controls.     

Gel-eluted liquid fraction entrapment electrophoresis: an electrophoretic method for broad molecular weight range proteome separation

Although well-established as a technique for protein purification, the application of continuous elution tube gel electrophoresis to proteome fractionation remains problematic. Difficulties associated with sample collection, particularly at the high mass range or at low sample loadings, continue to plague the technique. Furthermore, an upper mass limit is imposed as slow-moving higher molecular weight proteins are progressively diluted during the collection phase. In short, with current technology, effective separation over a broad mass range has not been achieved. In this work, we present improved techniques for continuous elution tube gel electrophoresis to accommodate broad mass range separation of proteins. Our device enables rapid partitioning of a proteome into discrete mass range fractions in the solution phase. High recovery is achieved at submicrogram to milligram sample loadings. We demonstrate comprehensive, reproducible separations of protein mixtures, as well as separation of a proteome in as fast as 1 h, over mass ranges from below 10 to 250 kDa. Finally, we identified proteins from a prefractionated standard protein mixture using liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis.     

Improved resolution of glycoproteins by chromatography with concanavalin A immobilized on microparticulate silica via temperature-programmed elution

The ability of the column temperature to control elution in the affinity chromatography of glycoproteins (e.g., ovalbumin and horseradish peroxidase) on silica immobilized concanavalin A has been studied. Column temperature programs can be achieved by placing a small HPLC column within a commercial mobile phase preheater assembly. It is shown that elution of adsorbed proteins can be initiated by changing the column temperature without altering the chemical composition of the mobile phase. Further, due to the enhancement in the rate of dissociation of the sample from the ligand, the peaks are narrowed. The resolution can be controlled by changing the initial temperature, dwell time at the initial temperature, and the rate of change of the temperature program. Addition of a competitive binding agent to the mobile phase decreases the temperature needed to elute strongly retained proteins. The effect of heating the column through many thermal cycles is assessed by periodically measuring the retention of a small monosaccharide that binds to the immobilized concanavalin A. The effect of two different immobilization procedures (glutaraldehyde and carbonyldiimidazole), as well as the effect of including a monosaccharide in the mobile phase, on the stability of the column is easily monitored by thermal elution chromatography. The effect of column temperature on the above glycoproteins has been assessed through studies of enzyme activities and anion exchange and isoelectric focusing patterns before and subsequent to temperature-programmed elution affinity chromatography.     

Determination of cabergoline by electrospray ionization tandem mass spectrometry: picogram detection via column focusing sample introduction.

An electrospray ionization tandem mass spectrometric method was developed for low-picogram detection of an ergot alkaloid, cabergoline, in coyote plasma extracts. Cabergoline is under investigation as an abortifacient in canid species. Central to the successful development of this method was the ability to introduce relatively large sample volumes into the mass spectrometer. This was achieved by focusing the analyte on a conventional high-performance liquid chromatography guard column prior to elution into the spectrometer. Volumes up to at least 900 microL could be injected onto the guard column using a 100% aqueous mobile phase. Cabergoline retained on the column was eluted as a discreet band into the mass spectrometer by the rapid addition of methanol (30%) to the mobile phase. As compared to flow injection sample introduction, the ability to inject larger sample volumes led to a greatly lowered detection limit. Using this technique and a modification of a previously reported extraction procedure, cabergoline could be determined in coyote plasma at concentrations as low as 9 pg of cabergoline/mL of plasma.     

Determination of naphthalene-2,3-dicarboxaldehyde-labeled amino acids by open tubular liquid chromatography with electrochemical detection

Naphthalene-2,3-dicarboxaldehyde (NDA) has been investigated as a new derivatizing reagent for the electrochemical detection of tagged amino acids. Gradient elution allowed for the separation of 18 NDA-derivatized amino acids on an open tubular liquid chromatography column in less than 50 min. Gradient elution and electrochemical detection were found to be compatible. A detection limit of 36 amol was obtained for the asparagine-NDA derivative. The usefulness of this technique for quantitation was demonstrated by the analysis of the NDA-tagged hydrolysis products from bovine chymotrypsinogen.     

Quantification in comprehensive two-dimensional liquid chromatography

Although the use of comprehensive two-dimensional liquid chromatography (LCxLC) as a powerful separation technique is continuously increasing, its employment in quantification experiments is rather limited. The present research is focused on the quantification of a series of standards, as well as real-world sample compounds, by using dedicated laboratory-constructed LCxLC software, developed through a novel approach. Moreover, the difficulties encountered during software operation, in various elution conditions, are described and discussed. The results attained were compared with those observed in conventional LC, and no statistically significant differences were observed in the determination of aurapten in grapefruit oil. However, a loss in sensitivity was observed when using LCxLC (limit of detection = 0.10 ppm) compared to conventional LC (limit of detection = 0.05 ppm) as a consequence of the sample dilution in comprehensive two-dimensional liquid chromatography.     

Determination of hydrolysis products of sulfur mustards by reversed-phase microcolumn liquid chromatography coupled on-line with sulfur flame photometric detection and electrospray ionization mass spectrometry using large-volume injections and peak compression

On-line coupling of reversed-phase microcolumn liquid chromatography (micro-RPLC) and sulfur-selective flame photometric detection (S-FPD) was studied for the selective and direct determination of thiodiglycol, bis(2-hydroxyethylthio)methane, 1,2-bis(2-hydroxyethylthio)ethane, 1,3-bis(2-hydroxyethylthio)propane, and 1,4-bis(2-hydroxyethylthio)butane, which are breakdown products of the chemical warfare agents called sulfur mustards. Both isocratic and gradient elution were used. To improve sensitivity, large-volume injections were applied together with peak compression by displacement for late-eluting analytes. With S-FPD, detection limits of 1 microgram/mL were obtained for all compounds. Using the same approach, the target analytes as well as various oxidation products could be identified by micro-RPLC with electrospray ionization mass spectrometry (ESI-MS) and ESI-MS/MS. The optimized micro-RPLC-S-FPD system was successfully used for the analysis of a spiked soil sample.     

Exploring the possibilities of cryogenic cooling in liquid chromatography for biological applications: a proof of principle

The possibilities to use cryogenic cooling to trap components in liquid chromatography was investigated. In a first step, van 't Hoff plots were measured with a reversed-phase column using the temperature control unit of a conventional high performance liquid chromatography (HPLC) system to gain insight in the retention behavior of proteins at low temperatures. It was estimated that retention factors in the range of k = 10(4) could be achieved at T = -20 °C for lysozyme, indicating that temperature is a usable parameter to trap components in LC. In a next step, trapping experiments were carried out on a nano-LC system, equipped with a UV-detector, using a commercial reversed-phase column. An in-house built setup, allowing cooling of a segment of the column down to temperatures below T = -20 °C, was used to trap components. Experiments were conducted under isocratic and gradient conditions with methanol as organic solvent. It is demonstrated that, by thermally trapping and elution of components, an enhanced S/N ratio and decreased peak widths can be obtained. At the same time, a significant increase in pressure drop occurs during the cooling process. Limitations and benefits of the technique are further discussed.     

Optimization of field-amplified sample injection for analysis of peptides by capillary electrophoresis-mass spectrometry

A versatile experimental approach is described to achieve very high sensitivity analysis of peptides by capillary electrophoresis-mass spectrometry with sheath flow configuration based on optimization of field-amplified sample injection. Compared to traditional hydrodynamic injection methods, signal enhancement in terms of detection sensitivity of the bioanalytes by more than 3000-fold can be achieved. The effects of injection conditions, composition of the acid and organic solvent in the sample solution, length of the water plug, sample injection time, and voltage on the efficiency of the sample stacking have been systematically investigated, with peptides in the low-nanomolar (10(-9) M) range readily detected under the optimized conditions. Linearity of the established stacking method was found to be excellent over 2 orders of magnitude of concentration. The method was further evaluated for the analysis of low concentration bioactive peptide mixtures and tryptic digests of proteins. A distinguishing feature of the described approach is that it can be employed directly for the analysis of low-abundance protein fragments generated by enzymatic digestion and a reversed-phase-based sample-desalting procedure. Thus, rapid identification of protein fragments as low-abundance analytes can be achieved with this new approach by comparison of the actual tandem mass spectra of selected peptides with the predicted fragmentation patterns using online database searching algorithms.     

Column precipitation chromatography: an approach to quantitative analysis of eigencolloids

A new column precipitation chromatography (CPC) technique, capable of quantitatively measuring technetium eigencolloids in aqueous solutions, is presented. The CPC technique is based on the destabilization and precipitation of eigencolloids by polycations in a confined matrix. Tc(IV) colloids can be quantitatively determined from their precipitation onto the CPC column (separation step) and their subsequent elution upon oxidation to pertechnetate by peroxide (elution step). A clean-bed particle removal model was used to explain the experimental results.     

Chelation ion chromatography as a method for trace elemental analysis in complex environmental and biological samples

The development and evaluation of a new method for the determination of trace transition and rare-earth elements based on the combination of chelation and ion chromatography are described. The new method, chelation ion chromatography (Chelation IC), uses a chelating column to concentrate and separate transition and rare-earth elements from the common alkali and alkaline-earth metals, as well as other matrix components, prior to analysis by ion chromatography. The sample fraction from the chelating column contains only the concentrated analyte ions, thus eliminating interfering matrix components from complex matrices such as seawater and digested biological, botanical, and geological materials. This combination of chelation and ion chromatography provides a technique that makes possible the determination of trace elements in complex matrices that have proven to be difficult or impossible to analyze by ion chromatography or conventional atomic spectroscopy techniques.     

High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics

We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi. With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency. To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations. Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.     

Targeted analyte detection by standard addition improves detection limits in matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) has proven an effective tool for fast and accurate determination of many molecules. However, the detector sensitivity and chemical noise compromise the detection of many invaluable low-abundance molecules from biological and clinical samples. To challenge this limitation, we developed a targeted analyte detection (TAD) technique. In TAD, the target analyte is selectively elevated by spiking a known amount of that analyte into the sample, thereby raising its concentration above the noise level, where we take advantage of the improved sensitivity to detect the presence of the endogenous analyte in the sample. We assessed TAD on three peptides in simple and complex background solutions with various exogenous analyte concentrations in two MALDI matrices. TAD successfully improved the limit of detection (LOD) of target analytes when the target peptides were added to the sample in a concentration close to optimum concentration. The optimum exogenous concentration was estimated through a quantitative method to be approximately equal to the original LOD for each target. Also, we showed that TAD could achieve LOD improvements on an average of 3-fold in a simple and 2-fold in a complex sample. TAD provides a straightforward assay to improve the LOD of generic target analytes without the need for costly hardware modifications.     

MALDI MS sample preparation by using paraffin wax film: systematic study and application for peptide analysis

Recently developed sample preparation techniques employing hydrophobic sample support have improved the detection sensitivity and mass spectral quality of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). These methods concentrate the samples on target by minimizing the sample area via the solvent repellent effect of the target surface. In the current study, we employed the use of paraffin wax film (Parafilm M) for improved MALDI MS analysis of low-abundance peptide mixtures, including neuronal tissue releasate and protein tryptic digests. This thin film was found to strongly repel polar solvents including water, methanol, and acetonitrile, which enabled the application of a wide range of sample preparation protocols that involved the use of various organic solvents. A "nanoliter-volume deposition" technique employing a capillary column has been used to produce tiny ( approximately 400 microm) matrix spots of 2,5-dihydroxybenzoic acid on the film. By systematically optimizing the sample volume, solvent composition, and film treatment, the Parafilm M substrate in combination with the nanoliter-volume matrix deposition method allowed dilute sample to be concentrated on the film for MALDI MS analysis. Peptide mixtures with nanomolar concentrations have been detected by MALDI time-of-flight and MALDI Fourier transform ion cyclotron resonance mass spectrometers. Overall, the use of Parafilm M enabled improved sensitivity and spectral quality for the analysis of complex peptide mixtures.     

高效液相色谱法测定食品中姜黄素

本文研究建立了食品中姜黄素的高效液相色谱测定方法。样品中的姜黄索经乙醇：氨水（3：1）提取后,聚酰胺吸附,再经甲醇氨水洗脱、浓缩,液相色谱二级管阵列检测器检测。姜黄索标准在0．5μg／mL-50μg／mL范围内具有良好的线性,该方法回收率高,样品定量检测限（LOQ）为0．2mg／kg,可作为糖果、糕点、面制品等食品的定量检测方法。