Elucidation of carotenoid patterns in citrus products by means of comprehensive normal-phase x reversed-phase liquid chromatography

A novel approach for carotenoid analysis has been developed. Orange essential oil and juice carotenoids were separated by means of comprehensive dual-gradient elution HPLC, using normal phase with a microbore silica column in the first dimension (first D), reversed phase with a monolithic C18 column in the second dimension (second D), and a 10-port switching valve as an interface. An on-line photodiode array detector was used in order to obtain absorption spectra. Peak identification was obtained by combining retention data with the UV-visible spectra.     

Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins

A comprehensive two-dimensional (2-D) liquid chromatographic separation system is presented. The system uses a microbore cation exchange column, operated under gradient conditions, as the first dimension separation. Effluent from this first column alternately fills one of two loops on a computer-controlled eight-port valve. A second pump then forces loop material onto a second column, a size exclusion column. UV detection is used, and the system is applied to the separation of protein standards and serum proteins. The 2-D system has a higher resolving power and peak capacity than either of the two columns used alone. The entire first column effluent is analyzed on the second column in virtually the same time it takes to complete the first column separation, without the use of stopped flow methods. The entire system is automated and operated under computer control. Three-dimensional (3-D) data representation provides a means of viewing peak profiles in either separation dimension and contour mapping of the 3-D data provides a more reliable means of peak identification from run to run than that provided by single-column elution times.     

An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation

A comprehensive on-line two-dimensional 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight below 20 kDa. The system setup provided fast separations and high resolving power and is considered to be a complementary technique to 2D gel electrophoresis in proteomics. The on-line system reproducibly resolved approximately 1000 peaks within the total analysis time of 96 min and avoided sample losses by off-line sample handling. The low-molecular-weight target analytes were separated from the matrix using novel silica-based restricted access materials (RAM) with ion exchange functionalities. The size-selective sample fractionation step was followed by anion or cation exchange chromatography as the first dimension. The separation mechanism in the subsequent second dimension employed hydrophobic interactions using short reversed-phase (RP) columns. A new column-switching technique, including four parallel reversed-phase columns, was employed in the second dimension for on-line fractionation and separation. Gradient elution and UV detection of two columns were performed simultaneously while loading the third and regenerating the fourth column. The total integrated workstation was operated in an unattended mode. Selected peaks were collected and analyzed off-line by MALDI-TOF mass spectrometry. The system was applied to protein mapping of biological samples of human hemofiltrate as well as of cell lysates originating from a human fetal fibroblast cell line, demonstrating it to be a viable alternative to 2D gel electrophoresis for mapping peptides and small proteins.     

Generating multiple independent retention index data in dual-secondary column comprehensive two-dimensional gas chromatography

A method producing simultaneously three retention indexes for compounds has been developed for comprehensive two-dimensional gas chromatography by using a dual secondary column approach (GC x 2GC). For this purpose, the primary flow of the first dimension column was equally diverted into two secondary microbore columns of identical geometry by means of a three-way flow splitter positioned after the longitudinally modulated cryogenic system. This configuration produced a pair of comprehensive two-dimensional chromatograms and generated retention data on three different stationary phases in a single run. First dimension retention indexes were determined on a polar SolGel-Wax column under linear programmed-temperature conditions according to the van den Dool approach using primary alcohol homologues as the reference scale. Calculation of pseudoisothermal retention indexes in both second dimensions was performed on low-polarity 5% phenyl equivalent polysilphenylene/siloxane (BPX5) and 14% cyanopropylphenyl/86% dimethylpolysiloxane (BP10) columns. To construct a retention correlation map in the second dimension separation space upon which KovAts indexes can be derived, two methods exploiting "isovolatility" relationships of alkanes were developed. The first involved 15 sequential headspace samplings of selected n-alkanes by solid-phase microextraction (SPME), with each sampling followed by their injection into the GC at predetermined times during the chromatographic run. The second method extended the second dimension retention map and consisted of repetitive introduction of SPME-sampled alkane mixtures at various isothermal conditions incremented over the temperature program range. Calculated second dimension retention indexes were compared with experimental values obtained in conventional one-dimensional GC. A case study mixture including 24 suspected allergens (i.e., fragrance ingredients) was used to demonstrate the feasibility and potential of retention index information in comprehensive 2D-GC.     

Ultra performance liquid chromatography isotope dilution tandem mass spectrometry for the absolute quantification of proteins and peptides

A selective, rapid, and sensitive 12.7-min ultra performance liquid chromatography-isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) method was developed and compared to conventional high-performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-ID/MS/MS) for the absolute quantitative determination of multiple proteins from complex matrixes. The UPLC analysis was carried out on an Acquity UPLC ethylene-bridged hybrid (BEH) C18 reversed-phase column (50 x 2.1 mm i.d., 1.7-microm particle size) with gradient elution at a flow rate of 300 microL/min. For the HPLC separation, a similar gradient profile on a reversed-phase C18 column with dimensions of 150 x 1.0 mm at a flow rate of 30 microL/min was utilized. The aqueous and organic mobile phases were 0.1% formic acid in water and acetonitrile, respectively. Detection was performed on a triple-quadrupole mass spectrometer operated in the multiple reaction monitoring mode. Linear calibration curves were obtained in the concentration range of 10-90 fmol/microL. Relative standard deviation values equal to or less than 6.5% were obtained by the UPLC-ID/MS/MS method, thus demonstrating performance equivalent to conventional HPLC-ID/MS/MS for isotope dilution quantification of peptides and proteins. UPLC provides additional dimensions of rapid analysis time and high-sample throughput, which expands laboratory emergency response capabilities over conventional HPLC.     

Capillary trap column with strong cation-exchange monolith for automated shotgun proteome analysis

A 150 microm internal diameter capillary monolithic column with a strong cation-exchange stationary phase was prepared by direct in situ polymerization of ethylene glycol methacrylate phosphate and bisacrylamide in a trinary porogenic solvent consisting dimethylsulfoxide, dodecanol, and N,N'-dimethylformamide. This phosphate monolithic column exhibits higher dynamic binding capacity, faster kinetic adsorption of peptides, and more than 10 times higher permeability than the column packed with commercially available strong cation-exchange particles. It was applied as a trap column in a nanoflow liquid chromatography-tandem mass spectrometry system for automated sample injection and online multidimensional separation. It was observed that the sample could be loaded at a flow rate as high as 40 microL/min with a back pressure of approximately 1300 psi and without compromising the separation efficiency. Because of its good orthogonality to the reversed phase separation mechanism, the phosphate monolithic trap column was coupled with a reversed-phase column for online multidimensional separation of 19 microg of the tryptic digest of yeast proteins. A total of 1522 distinct proteins were identified from 5608 unique peptides (total of 54,780 peptides) at the false positive rate only 0.46%.     

Simple and comprehensive two-dimensional reversed-phase HPLC using monolithic silica columns

Simple and comprehensive two-dimensional (2D)-HPLC was studied in a reversed-phase mode using monolithic silica columns for second-dimension (2nd-D) separation. Every fraction from the first column, 15 cm long (4.6-mm i.d.), packed with fluoroalkylsilyl-bonded (FR) silica particles, was subjected to the separation in the 2nd-D using one or two octadecylsilylated (C(18)) monolithic silica columns (4.6-mm i.d., 3 cm). Monolithic silica columns in the 2nd-D were eluted at a flow rate of up to 10 mL/min with separation time of 30 s that meets the fractionation every 15-30 s at the first dimension (1st-D) operated at a flow rate of 0.4-0.8 mL/min. Three cases were studied. (1) In the simplest scheme of 2D-HPLC, effluent of the 1st-D was directly loaded into an injector loop of 2nd-D HPLC for 28 s, and 2 s was allowed for injection. (2) Two six-port valves each having a sample loop were used to hold the effluent of the 1st-D alternately for 30 s for one 2nd-D column to effect comprehensive 2D-HPLC without the loss of 1st-D effluent. (3) Two monolithic silica columns were used for 2nd-D by using a switching valve and two sets of 2nd-D chromatographs separating each fraction of the 1st-D effluent with the two 2nd-D columns alternately. In this case, two columns of the same stationary phase (C(18)) or different phases, C(18) and (pentabromobenzyloxy)propylsilyl-bonded (PBB), could be employed at the 2nd-D, although the latter needed two complementary runs. The systems produced peak capacity of approximately 1000 in approximately 60 min in cases 1 and 2 and in approximately 30 min in case 3. The three stationary phases, FR, C(18), and PBB, showed widely different selectivity from each other, making 2D separations possible. The simple and comprehensive 2D-HPLC utilizes the stability and high efficiency at high linear velocities of monolithic silica columns.     

High-performance polymer-based monolithic capillary column

In this report, we introduce a new entry of high-performance polymer-based monolithic capillary column for mainly small molecules. This capillary column was prepared using a newly introduced epoxy monomer with diamines. Simply heat-induced polycondensation in an appropriate porogenic solvent afforded a really homogeneous co-continuous monolithic structure having submicrometer-size skeletons with micrometer-size through-pores. We were also able to prepare chiral monolithic columns using a chiral epoxy monomer as well as a chiral diamine. A 21.5-cm-long, 100-mum-i.d. column afforded up to 40 000 theoretical plate numbers (N) for alkylbenzenes in 60% aqueous acetonitrile as a reversed-phase-mode stationary phase. Due to a quite low column pressure drop, a 150-cm-long column was prepared. This long column afforded up to 200 000 plates for alkylbenzenes with only a 4-MPa column pressure drop. In contrast, in 100% acetonitrile, this column has "HILIC" property to show up to 60 000 plates for methanol with a 17.5-cm-long column. In this mode, we were able to separate nucleic acids. In addition, we have prepared a chiral column with both of the chiral epoxy monomers and an amine. This column was able to chirally discriminate a racemic alcohol in a reversed-phase mode.     

Preparation and application of methacrylate-based cation-exchange monolithic columns for capillary ion chromatography

Polymer-based strong cation-exchange monolithic capillary columns with different capacities were constructed for ion chromatography by radical polymerization of glycidyl methacrylate (GMA) and ethylene dimethacrylate in a 250-microm-i.d. fused-silica capillary and its subsequent sulfonation based on ring opening of epoxides with 1 M Na(2)SO(3). The cation-exchange capacities can easily and reproducibly be controlled in the range of up to 300 microequiv/mL by changing the immersion time of the epoxy-containing polymer in the Na(2)SO(3) solution. The chromatographic performance of the produced monolithic capillary columns was evaluated through the separation of a model mixture of common cations such as Na(+), NH(4)(+), K(+), Mg(2+), and Ca(2+). As an example, these cations could be well separated from one another on a 15-cm-long cation-exchange monolithic column (column volume, 7.4 microL) with a capacity of 150 microequiv/mL by elution with 10 mM CuSO(4). The pressure drop of this 15-cm column was approximately 1 MPa at a normal linear velocity of 1 mm/s (a flow rate of 3 microL/min), and the numbers of theoretical plates for the cations were above 3000 plates/15 cm. This GMA-based cation-exchange monolithic column could withstand high linear velocities of at least 10 mm/s. Over a period of at least two weeks of continuous use, no significant changes in the selectivity and resolution were observed. The applicability of a flow rate gradient elution and the feasibility of direct injection determination of major cations in human saliva sample were also presented.     

Isoelectric focusing nonporous RP HPLC: a two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry

A novel two-dimensional liquid-phase separation method was developed that is capable of resolving large numbers of cellular proteins. The proteins are separated by pI using isoelectric focusing in the first dimension and by hydrophobicity using nonporous reversed-phase HPLC in the second dimension (IEF-NP RP HPLC). Proteins were mapped using original software in order to create a protein pattern analogous to that of the 2-D PAGE image. RP HPLC peaks are represented by bands of different intensity in the 2-D image, according to the intensity of the peaks eluting from the HPLC. Each peak was collected as the eluent of the HPLC separation in the liquid phase. The proteins collected were identified using proteolytic enzymes, MALDI-TOF MS and MSFit database searching. Using IEF-NP RP HPLC, approximately 700 bands were resolved in a pI range from 3.2 to 9.5 and 38 different proteins with molecular weights ranging from 12,000 to 75,000 were identified. In comparison to a 2-D gel separation of the same human erythroleukemia cell line lysate, the IEF-NP RP HPLC produced improved resolution of low mass and basic proteins. In addition, the proteins remained in the liquid phase throughout the separation, thus making the entire procedure highly amenable to automation and high throughput. It is demonstrated that IEF-NP RP HPLC provides a viable alternative to the 2-D gel separation method for the screening of protein profiles.     

Performance of a monolithic silica column in a capillary under pressure-driven and electrodriven conditions

A continuous macroporous silica gel network was prepared in a fused-silica capillary and evaluated in reversed-phase liquid chromatography. Under pressure-driven conditions, the monolithic silica column derivatized to C18 phase (100 microns in diameter, 25 cm in length, silica skeleton size of approximately 2.2 microns) produced plate heights of about 23 and 81 microns at 0.5 mm/s with a pressure drop of 0.4 kg/cm2, and at 4.0 mm/s with 3.6 kg/cm2, respectively, in 90% acetonitrile for hexylbenzene with a k value of 0.7. The separation impedance, E, calculated for the present monolithic silica column was much smaller at a low flow rate than those for particle-packed columns, although higher E values were obtained at a higher flow rate. Considerable dependence of column efficiency on the linear velocity of the mobile phase was observed despite the small size of the silica skeletons. A major source of band broadening in the HPLC mode was found in the A term of the van Deemter equation. The performance of the continuous silica capillary column in the electrodriven mode was much better than that in the pressure-driven mode. Plate heights of 7-8 microns were obtained for alkylbenzenes at 0.7-1.3 mm/s, although the electroosmotic flow was slow. In HPLC and CEC mode, the dependency of plate height on k values of the solutes was observed as seen in open tube chromatography presumably due to the contribution of the large through-pores. Since monolithic silica capillary columns can provide high permeability, the pressure-driven operation at a very low pressure can afford a separation speed similar to CEC at a high electric field.     

Impulse-driven heated-droplet deposition interface for capillary and microbore LC-MALDI MS and MS/MS

An automated off-line liquid chromatography-matrix-assisted laser desorption ionization (LC-MALDI) interface capable of coupling both capillary and microbore LC separations with MALDI mass spectrometry (MS) and tandem mass spectrometry (MS/MS) has been developed. The interface is a combination of two concepts: analyte concentration from heated hanging droplets and impulse-driven droplet deposition of LC fractions onto a MALDI sample plate. At room temperature the interface allows the coupling of capillary LC separations (i.e., flow rate of <5 microL/min) with MALDI MS. With heating, it can be used to combine microbore LC operated at a relatively high flow rate of up to 50 microL/min with MALDI MS. The collected fractions can be analyzed by MALDI MS and MS/MS instruments, such as time-of-flight (TOF) and quadrupole-TOF MS. Performance of the interface was examined using several peptide and protein standards. It was shown that, using MALDI-TOF MS, [GLU1]-fibrinopeptide B could be detected with a total injection amount of 5 fmol to microbore LC. Chromatographic performance was also monitored. A peak width of 12 s at half-height for [GLU1]-fibrinopeptide B showed no evidence of band broadening due to the interface. The ability of the interface to mitigate ion suppression was studied using a mixture of 100 fmol of [GLU1]-fibrinopeptide B and 10 pmol of cytochrome c tryptic digest. Although fully suppressed under direct MALDI conditions, LC-MALDI analysis was able to detect the 100 fmol peptide with 10 s fraction collection. Finally, the ability to inject relatively large sample amounts to improve detectability of low-abundance peptides was illustrated in the analysis of phosphopeptides from alpha-casein tryptic digests. A digest loaded on column to 2.4 microg and analyzed by LC-MALDI MS/MS resulted in 82% sequence coverage and detection of all nine phosphoserine residues. It is concluded that, being able to handle both high- and low-flow LC separations, the impulse-driven heated-droplet interface provides the flexibility to carry out MALDI analysis of peptides and proteins depending on the information sought after, analysis speed, and sample size.     

Performance of monolithic silica capillary columns with increased phase ratios and small-sized domains

Monolithic silica capillary columns for HPLC were prepared from tetramethoxysilane to have smaller sized domains and increased phase ratios as compared to previous materials, and their performance was evaluated. The monolithic silica columns possessed an external porosity of 0.65-0.76 and a total porosity of 0.92-0.95 and showed considerably higher performance and greater retention factors in a reversed-phase mode after chemical modification than columns previously reported. An octadecylsilylated monolithic silica column with the smallest domain size (through-pores of approximately 1.3 microm and silica skeletons of approximately 0.9 microm) showed a plate height of less than 5 microm at optimum linear velocities (u) of 2-3 mm/s in 80% acetonitrile for a solute having retention factors of approximately 1, and approximately 7 microm at u = 8 mm/s. With a permeability similar to that of a column packed with 5-microm particles, the monolithic silica columns were able to attain column efficiencies comparable to that of particulate columns packed with 2-2.5-microm particles, and showed performance in the "forbidden region" for the previous columns. The performance of the monolithic column can be compared favorably with that of a particle-packed column when 15,000-30,000 or more theoretical plates are desired at a pressure drop of 20-40 MPa or lower. The increased homogeneity of the co-continuous structures, in addition to the small-sized domains, contributed to the higher performance as compared to previous monolithic silica columns.     

Two-dimensional separation method for analysis of Bacillus subtilis metabolites via hyphenation of micro-liquid chromatography and capillary electrophoresis

A novel two-dimensional separation method, which hyphenated chromatography and electrophoresis, was developed for analysis of Bacillus subtilis metabolites. Micro-liquid chromatography (LC) with a monolithic silica-ODS column was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Concentration strategies, namely, dynamic pH junction and sweeping, were selectively employed to interface the two dimensions, which proved to be beneficial for the detection of metabolites. For system evaluation, an artificial sample containing 54 standard metabolites was separated according to their hydrophobicity by micro-LC with gradient mode. The early-eluting fractions were separated by capillary zone electrophoresis in combination with dynamic pH junction, while the late-eluting fractions were separated by sweeping micellar electrokinetic chromatography. The middle fractions were analyzed by both modes of CE. Under the optimum conditions, all the components in the artificial sample could be well resolved. The method was applied to profile B. subtilis metabolites. Some crucial metabolites were identified. This method provided great potential for resolving complex biological samples containing compounds having different characteristics.     

Automated ultra-high-pressure multidimensional protein identification technology (UHP-MudPIT) for improved peptide identification of proteomic samples

Multidimensional separation is one of the most successful approaches for proteomics studies that deal with complex samples. We have developed an automated ultra-high-pressure multidimensional liquid chromatography system that operates up to approximately 20 kpsi to improve separations and increase protein coverage from limited amount of samples. The reversed-phase gradient is operated in the constant-flow mode opposed to the constant-pressure mode, which is typical of previous ultra-high-pressure systems. In contrast to constant-pressure systems, the gradient shape is fully controllable and can be optimized for the type of samples to be run. The system also features fast sample loading/desalting using a vented column approach to improve sample throughput. This approach was validated on a soluble fraction from yeast lysate where we achieved approximately 30% more protein identifications using a 60-cm-long triphasic capillary column than with our traditional approach. Advantages of the use of a relatively long reversed-phase column (approximately 50 cm) for MudPIT-type experiments are also discussed.     

Characterization of capillary-channeled polymer fiber stationary phases for high-performance liquid chromatography protein separations: Comparative analysis with a packed-bed column

Capillary-channeled polymer (C-CP) fibers are investigated as reversed-phase (RP) stationary phases for high-performance liquid chromatography of proteins. A comparative analysis of column characteristics for polypropylene and poly(ethylene terephthalate) C-CP fiber columns and a conventional packed-bed (C4-derivatized silica) column has been undertaken. Five proteins (ribonuclease A, cytochrome c, lysozyme, myoglobin, bovine serum albumin) were used to investigate the separation characteristics under typical RP gradient conditions. Column performance was compared under standard (identical) and optimized RP chromatographic conditions. The gradient compositions utilized with the C-CP fiber columns are similar to those used with conventional columns, employing flow rates in the 1-6 mL/min range and gradient rates of approximately 1%/min. The packed-bed column was operated as prescribed by the column manufacturer. The retention factor (k'), separation factor (alpha), resolution (Rs), asymmetry factor (As), elution order, and peak capacity values of a four protein separations performed on the C-CP fiber columns are compared to the same separation on the C4 column. One unique feature observed here is the lessening of the percentage of organic modifier necessary to elute the proteins from the fiber phases with increased linear velocity. The potential contribution of the different stationary phases to protein denaturation was evaluated through a spectrophotometric enzymatic activity assay. The repeatability of retention times under both sets of conditions for six consecutive injections of lysozyme on each C-CP fiber column is < or =1.5% RSD. The column-to-column reproducibility of retention times for three columns of each fiber type is also < or =1.5% RSD. The overall performance of the C-CP fiber columns was comparable to the conventional column used in these studies. Basic characteristics demonstrated here suggested further developments in the areas of ultrafast protein separations and preparative-scale protein chromatography.     

Surface-alkylated polystyrene monolithic columns for peptide analysis in capillary liquid chromatography-electrospray ionization mass spectrometry

Macroporous poly(styrene-divinylbenzene) (PS-DVB) monoliths were prepared by in situ polymerization in PEEK, fused silica, or stainless steel tubing having an inner diameter of 75 or 125 microm. A process is described for subsequent alkylation of the flow-contacting surfaces of the monoliths. The process treats all the surfaces including through-pore surfaces of the rigid macroporous monolith with a solution containing a dissolved Friedel-Crafts catalyst, an alkyl halide (1-chlorooctadecane), and an organic solvent. This process produces an improved reversed-phase liquid chromatographic separation of peptides compared to an unmodified monolithic PS-DVB column. The surface octadecylation is not necessary for a reversed-phase separation of proteins since both unmodified and modified columns provide comparable results. Tryptic protein digests, standard proteins, and standard peptides were used to evaluate the monolithic columns by employing electrospray mass spectrometry detection. Potential applications in proteomics studies by mass spectrometry, which use the alkylated monolithic column engaged onto the nanofabricated electrospray ionization chip, are also discussed.     

Pesticide residue analysis by off-line SPE and on-line reversed-phase LC-GC using the through-oven-transfer adsorption/desorption interface

A new method to determine pesticide residue in water is presented. The described method includes using off-line solid-phase extraction (SPE) and on-line reversed-phase liquid chromatography-gas chromatography (RPLC-GC). An interface, based on a modified programmed temperature vaporizer (PTV) injector, packed with a suitable trapping material, is used for on-line RPLC-GC. The changes made in the PTV injector affect the pneumatic system, sample introduction, and solvent elimination. The new interface is easily capable of automation. Methanol/wate (70/30) is used as the eluent in the LC preseparation step. The LC column flow during elution is different from the flow during the transfer step. The transferred volumes range from 500 to 1400 microL (volume of the fractions of interest). Solvent elimination is almost 100% before the sample reaches the GC column. The described system does not show any variation of the peak retention times. The detection limit for real samples ranges from 0.04 to 1.5 ng/L, using NP detection.     

Universal two-dimensional HPLC technique for the chemical analysis of complex surfactant mixtures

Two-dimensional HPLC was applied for the first time to the analysis of complex surfactant mixtures. In the first dimension, ion chromatographic-type separations were performed on a diol column eluted by an acetonitrile-water (0.1% trifluoroacetic acid) gradient. Using this new technique, cationic and amphoteric surfactants were not retained at all, nonionic surfactants exhibited a weak and essentially unspecific retention, and anionic surfactants were retained mainly according to their functional group. Rather than detecting the analytes immediately after this first separation, successive fractions were automatically and quantitatively transferred to parallel C2 (dimethyl) and C4 (butyl) reversed-phase columns using an innovative setup. The second dimension of the separation then took place, by which the analytes were separated according to their hydrophobicity. Surfactants from all four classes, cationic, amphoteric, nonionic, and anionic, were separated simultaneously in single 54-min two-dimensional HPLC runs. The suitability of the method for quantitative measurements was demonstrated.     

Utility of postcolumn addition of 2-(2-methoxyethoxy)ethanol, a signal-enhancing modifier, for metabolite screening with liquid chromatography and negative ion electrospray ionization mass spectrometry

A strategy for highly sensitive metabolite screening by liquid chromatography-electrospray ionization (ESI) mass spectrometry with the negative-ion mode that involves the use of a reversed-phase column in gradient-elution mode and postcolumn addition of 2-(2-methoxyethoxy)ethanol (2-MEE), a novel signal-enhancing modifier, has been described. When a mobile phase of 50 mM ammonium acetate/acetic acid buffer (pH 4.4) at a flow rate of 100 microL/min was employed, poor ESI response of ibuprofen as a model drug, probably due to both the high surface tension of the mobile phase and the ion-suppression effect of acetate anion in the mobile phase, was observed. On the other hand, the postcolumn addition of 2-MEE (50 microL/min) into the mobile phase counteracted the ion suppression as well as the surface tension problem, resulting in approximately 100-fold signal enhancement of the analyte. The metabolite screening of ibuprofen in human urine was subsequently carried out comparing the results with and without postcolumn addition of 2-MEE. The results indicated that the postcolumn addition of 2-MEE dramatically improved the ESI responses of all urinary metabolites detected without affecting the chromatographic separation.