Protocol for liquid chromatography/mass spectrometry of glutathione conjugates using postcolumn solvent modification

A novel protocol for thermospray liquid chromatography/mass spectrometry (LC/MS) analysis of mixtures of glutathione conjugates is reported. Solvent conditions for optimal high-performance liquid chromatography are not always the same as for optimal thermospray ionization mass spectrometry. Labile glutathione conjugates that give poor spectra in aqueous ammonium acetate yield more intense molecular ion signals with increased percentages of acetonitrile. Direct injection thermospray ionization using 30-60% acetonitrile in aqueous ammonium acetate produced protonated molecular ions for glutathione conjugates of menadione, styrene oxide, pentachlorophenyl methyl sulfone, chlorodinitrobenzene, and chlorambucil. Since, the high percentages of organic modifier needed for good molecular ion intensity preclude chromatographic separation of these polar compounds, successful graphic separation of these polar compounds, successful LC/MS was facilitated by postcolumn addition of organic modifiers to the mobile phase. This new methodology allowed excellent chromatographic separations and thermospray ionization mass spectra to be obtained for a mixture of haloalkane glutathione conjugates. Moreover, cleavage of the gamma-glutamyl-cysteine amide bond of glutathione results in class-characteristic fragment ions. Changes in the fragmentation pathways in spectra acquired with and without organic modifiers shed light on the importance of the desolvation process in obtaining good molecular ion sensitivity in thermospray.     

Protein characterization by on-line capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry

Two-dimensional polyacrylmide gel electrophoresis (2D-PAGE), perhaps the most widely used method in proteomics research, is often limited by sensitivity and throughput. Capillary isoelectric focusing (CIEF) coupled with electrospray ionization (ESI) mass spectrometry (MS) provides a liquid-based alternative to 2D-PAGE that can overcome these problems but is limited by ampholyte interference and signal quenching in ESI-MS. Inserting a reversed-phase liquid chromatography (RPLC) step between CIEF and MS can remove this interference. In this work, a CIEF-RPLC-MS system is described for separation and characterization of proteins in complex mixtures. CIEF is performed with a microdialysis membrane-based cathodic cell that also permits protein fractions to be collected, washed to remove ampholyte, and analyzed by RPLC-MS. CIEF performance with this cell is equivalent to that achieved with a conventional cathodic cell, and no loss of protein is observed during faction collection. The cell can be easily and safely retrofitted into commercial instrumentation and is applicable for peptide analysis as well. Protein detection at the low-femtomole level is demonstrated with little or no interference from ampholyte, and CIEF-RPLC-MS data are used to construct a plot of pI vs MW for a protein mixture. The current instrumental configuration allows seven fractions in the pI range 3-10 to be analyzed by RPLC-MS in 2 h.     

Determination of peroxide-based explosives using liquid chromatography with on-line infrared detection

A nondestructive analytical method for peroxide-based explosives determination in solid samples is described. Reversed-phase high-performance liquid chromatography in combination with on-line Fourier transform infrared (FT-IR) detection is used for the analysis of triacetonetriperoxide (TATP) and hexamethylenetriperoxide diamine (HMTD). In contrast to other liquid chromatographic methods with optical detection, no derivatization or decomposition of the peroxides is required. The peroxides are identified and quantified via their characteristic absorption spectra in the mid-infrared range of the electromagnetic spectrum. The detection limit of 0.5 mmol L-1 for HMTD and 1 mmol L-1 for TATP allows the identification of the explosives in complex matrixes.     

Ultra-sensitive quantification of corticosteroids in plasma samples using selective solid-phase extraction and reversed-phase capillary high-performance liquid chromatography/tandem mass spectrometry

Low-dose corticosteroids may provide a favorable benefit/risk ratio for many therapeutic applications. However, the extremely low plasma drug concentrations achieved, in conjunction with the insufficient sensitivity/ selectivity of current analytical methods, renders the evaluation of corticosteroid pharmacokinetics (PK) a significant challenge under such conditions. Furthermore, targeted therapeutic strategies involving administration by inhalation or intraocular injection could result in very low but sustained systemic corticosteroid concentrations, which must be quantified to determine potential side effects. Here we describe a robust method for the ultrasensitive quantification of corticosteroids in plasma samples. This was achieved by the combination of a selective solid-phase extraction (SPE) with a highly sensitive capillary LC (microLC)-MS/MS analysis. SPE washing and elution conditions were optimized so that target drugs are selectively extracted from plasma. By eliminating most undesirable compounds from the sample matrix, this selective SPE procedure enabled a high sample loading volume on the microLC column without compromising chromatographic performance and operational robustness and helped to achieve ultralow detection limits for the corticosteroids in plasma. The effect of microLC separation on the signal-to-noise ratio of corticosteroid peaks in plasma samples was investigated. It was found that with sufficient microLC separation, sensitivity was improved because of a decrease in matrix effects and the removal of endogenous interferences. Detection limits of four clinically important corticosteroids (budesonide, dexamethasone, triamcinolone acetonide, and dexamethasone acetate) ranged from 0.2 to 1 pg/mL in plasma, and linearity was good for all drugs in the range of 5-5000 pg/mL. Accuracy was 88-107% and the variation (CV%) was 2.3-11.1%. A limit of quantification (LOQ) of 5 pg/mL was validated for all four compounds. We applied this method to quantify the low levels of triamcinolone acetonide (TACA) in porcine plasma following suprachoroidal administration, which is necessary to estimate systemic drug exposure resulting from this novel clinical approach for treating inflammatory diseases of the eye. TACA in plasma could be quantified at low pg/mL levels for up to 90 days posttreatment. To our knowledge, this is the first practical analytical approach that can monitor plasma corticosteroids after intraocular administration, given the ultralow plasma concentrations achieved. In summary, this strategy enables PK analysis of corticosteroids in treatment regimens that result in extremely low systemic concentrations, and the approach can be extended for the sensitive quantification of other drugs.     

Simultaneous enhancement of separation selectivity and solvent strength in reversed-phase liquid chromatography using micelles in hydro-organic solvents

The role of micelles and organic solvents as the modifiers of the aqueous mobile phase in reversed-phase liquid chromatography (RPLC) in controlling retention and selectivity is discussed. Elution strength increases in RPLC with an increase in organic solvent or micelle concentration. Simultaneous enhancement of separation selectivity with elution strength in the hybrid eluents of water-organic solvent-micelles was observed. This selectivity enhancement occurs systematically, i.e. peak separation increases monotonically with volume fraction of organic solvent added to micellar eluent, and is observed for a large number of ionic and nonionic compounds with different functional groups and for two surfactants (anionic and cationic). For two test mixtures, 13 amino acids/peptides and 15 phenols, it is shown that a better separation and shorter analysis time are observed at stronger hybrid eluents. This selectivity enhancement can be attributed to the competing partitioning equilibria in micellar LC systems and/or to the unique characteristics of micelles to compartmentalize solutes and organic solvents.     

Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.     

Discrimination of soil-borne fungi using fourier transform infrared attenuated total reflection spectroscopy

Fourier transform infrared (FT-IR) attenuated total reflection (ATR) spectroscopy was used to discriminate five commonly encountered soil-borne fungi that cause severe economic damage to agriculture: Colletotrichum, Fusarium, Pythium, Rhizoctonia, and Verticillium. Contrary to previous studies related to microorganism discrimination using FT-IR-ATR spectroscopy, the pathogen samples were not dried on the ATR crystal, which is a time-consuming operation. Rather, after removing some pathogen filaments from the solution using tweezers, these were placed directly on a flat ATR crystal and pressure was applied using a pressure clamp. Following water subtraction, baseline correction, and normalization of the spectra, principal component analysis was used as a data-reduction step and canonical variate analysis was used for discrimination. Discrimination was performed at the genus level and at the strain level for Colletotrichum. For discrimination between the five fungi at the genus level, the success rate for the validation samples ranged from 75% to 89%. For discrimination between the two Colletotrichum strains, the success rate was 78%. Comparison with spectra of similar fungi dried on the ATR crystal showed that both types of spectra were very similar, indicating that drying the samples on the ATR crystal is not required and can be replaced by mathematical post-processing of the spectra. For routine analyses that involve rapid screening of very large amounts of samples, this approach allows for increasing significantly the number of samples that can be analyzed daily.     

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.     

High-throughput proteomics using high-efficiency multiple-capillary liquid chromatography with on-line high-performance ESI FTICR mass spectrometry

We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10,000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for "backup" operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of approximately 650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of > 6 x 10(7) components. For yeast cytosolic tryptic digests > 100,000 polypeptides were detected, and approximately 1,000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (approximately 1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.     

Applications of Hadamard transform to gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry

Successful application of the Hadamard transform (HT) technique to gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) is described. Novel sample injection devices were developed to achieve multiple sample injections in both GC and LC instruments. Air pressure was controlled by an electromagnetic valve in GC, while a syringe pump and Tee connector were employed for the injection device in LC. Two well-known, abused drugs, 3,4-methylenedioxy-N-methylamphetamine (MDMA) and N, N-dimethyltryptamine (DMT), were employed as model samples. Both of the injection devices permitted precise successive injections, resulting in clearly modulated chromatograms encoded by Hadamard matrices. After inverse Hadamard transformation of the encoded chromatogram, the signal-to-noise (S/N) ratios of the signals were substantially improved compared with those expected from theoretical values. The S/N ratios were enhanced approximately 10-fold in HT-GC/MS and 6.8 in HT-LC/MS, using the matrices of 1023 and 511, respectively. The HT-GC/MS was successfully applied to the determination of MDMA in the urine sample of a suspect.     

Direct plasma analysis of drug compounds using monolithic column liquid chromatography and tandem mass spectrometry

A monolithic silica column high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed for the high-speed direct simultaneous determination of a drug discovery compound and its major circulating metabolite (M-72) in rat plasma. This methodology makes use of flow programming and an alkyl-bonded silica rod column for fast macromolecule removal and chromatographic separation without the need for significant sample preparation. The matrix ionization suppression effect on the monolithic column HPLC-MS/MS system was investigated using the postcolumn infusion technique. After 200 plasma injections on a 50 x 4.6 mm monolithic silica column, consistent column efficiency of close to 39,000 theoretical plates/m and reproducible retention times for the analytes were observed. The apparent on-column recoveries of 12 test compounds in rat plasma samples were greater than 90%. The proposed fast direct plasma injection method was tested over a 3-day period with the interday coefficient of variation less than 15% for both analytes.     

Determination of phospholipids from pulmonary surfactant using an on-line coupled silica/reversed-phase high-performance liquid chromatography system.

A basic normal-phase HPLC separation of phospholipids can be improved by introducing a limited contribution of solvophobic retention. For this purpose, the effect of an additional alkylsilica (C18) column of variable length coupled in series with a silica column was investigated. With increasing percentage of reversed phase in this system, the retention of phosphatidylglycerol increased. Phosphatidylinositol and phosphatidylserine were separated into molecular species. The "selective retention" defined in this study permits an evaluation of the solvophobic retention of phospholipids in the coupled system. An alternative column switching procedure is used for specific applications of the biphasic separation on chosen phospholipids. With this system, determination of phosphatidylglycerol and six other phospholipids from pulmonary surfactant could be performed.     

Hot phosphate-buffered water extraction coupled on-line with liquid chromatography/mass spectrometry for analyzing contaminants in soil

We evaluated the feasibility of analyzing rapidly traces of polar and medium polar contaminants in soil by coupling on-line a hot phosphate-buffered water extraction apparatus to a liquid chromatography/mass spectrometer system. Coupling was accomplished by using a small C-18 sorbent trap for collecting analytes and two six-port valves. The efficiency of this device was evaluated by extracting 13 selected pesticides from 200 mg of laboratory-aged soils by varying the extraction temperature, the extractant volume, and the flow rate at which the extractant passed through the extraction cell and the sorbent trap. In terms of extraction efficiency, robustness of the method, and extraction time, the best compromise was that of using 8 mL of extractant at 90 degrees C and 0.5 mL/min flow rate. Under these conditions, recoveries of 11 out of 13 analytes ranged between 82 and 103%, while those of the least hydrophilic pesticides, i.e., neburon and prochloraz, were 73 and 63%, respectively. By increasing the extractant volume to 60 mL, additional amounts of the two latter compounds could be recovered. Under this condition, however, the most hydrophilic analytes were in part no more retained by the C-18 sorbent trap. From a naturally 1.5-year aged soil, hot phosphate-buffered water removed larger amounts of three herbicides and hydroxyterbuthylazine (a terbuthylazine degradation product) than pure water and Soxhlet extraction. This result seems to confirm that hot phosphate buffer is also able to remove from soil those fractions of contaminants that, on aging, are sequestered into the humic acid framework.     

Quantification of volatile organics in soil aging experiments using fourier transform infrared spectroscopy

On-line Fourier transform infrared (FT-IR) spectroscopy was applied to monitor the concentration of halogenated volatile organic compounds in a sample-preparation process that simulates long-term, slow accumulation of contaminants in soils (i.e., aging). Artificial aging is conducted by circulating a supercritical fluid solution containing the contaminant(s) of interest through a packed soil column. Mid-infrared spectra of several volatile halocarbons were measured in supercritical Xe and CO(2) to evaluate possible interferences from the strong absorption of CO(2). Although some of the C-X bands were partially masked in supercritical CO(2), all of the compounds studied had distinct spectral features in the region 1400-700 cm(-1) and could be monitored in either solvent. Quantitative measurements of halogenated volatile organics in supercritical CO(2) were demonstrated with CCl(4). Excellent results were obtained over the range 7-280 mM. Representative artificial aging experiments were conducted on two test soils using CCl(4) as the contaminant. On-line (FT-IR) estimates of the aged soil concentrations were 1.3-4.4 times higher than off-line concentrations obtained by gas chromatography/mass spectrometry. The discrepancies were primarily ascribed to post-aging losses that occurred during depressurization and subsequent sample handling. FT-IR spectroscopy is shown to be a powerful tool for monitoring soil loading behavior and for developing artificial aging protocols.     

人血清中血糖的近红外光谱快速检测

应用傅利叶变换近红外光谱透射技术结合偏最小二乘法 ( PLS) ,快速定量分析了人血清中血糖含量 .利用内部交叉验证和自动优化功能对预测模型进行了优化 ,确定了最优建模参数 .模型对人血清中葡萄糖定标样品集的实测含量与预测含量的相关系数 r=0 .91 48,内部校正均方差 RMSECV=0 .487mmol/L.     

Determination of mycophenolic acid and mycophenolate mofetil by high-performance liquid chromatography using postcolumn derivatization

An efficient method to lower the optical detection limit is described using the displacement of an absorption and emission band of an analyte after a polarity change in different solvents. This solvatochromic effect was used in a RP-HPLC assay for the fluorescence detection of mycophenolic acid (6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-5-phthalanyl)-4-methyl-4-hexenoic acid, MPA) and the prodrug mycophenolate mofetil (MMF), the N-(2-hydroxyethyl)morpholino ester of MPA. The rational to use fluorescence detection is based on the behavior of MMF and MPA, which fluoresce in a basic medium (pH >9.5). Following a simple protein precipitation, the analytes were separated in an isocratic RP-HPLC system. The postcolumn generation of the phenolate anions of MPA and MMF was achieved by addition of an aqueous sodium hydroxide solution regulated by a newly developed continuous-flow liquid control system. MPAG, not directly accessible for fluorescence detection, was analyzed after enzymatic deglucuronidation to MPA. Compared to published quantification limits for MPA and MMF by UV detection, this method is more than 100-fold more sensitive, with a lower limit of quantification of 45 fmol for both MPA and MMF.     

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.