A miniature mass spectrometer for liquid chromatography applications

A miniature mass spectrometer capable of detecting analytes eluting from a high-performance liquid chromatography (HPLC) system is described and demonstrated for the first time. The entire instrument, including all pumps and the computer, is contained within a single enclosure that may be conveniently accommodated at the base of the HPLC stack. The microspray ion source, vacuum interface, ion guide, and quadrupole ion filter are all microengineered. These components are fabricated in batches using microelectromechanical systems (MEMS) techniques and considered to be consumables. When coupled to a standard HPLC system using an integrated passive split, the limit of detection for reserpine while scanning the full mass range is 5 ng on-column (1 pg of which is passed to the microspray). The mass range is m/z 100-800, and each spectrum is typically acquired at a rate of 1 scan per second.     

A new concept for isotope ratio monitoring liquid chromatography/mass spectrometry

A new interface for the on-line coupling of a liquid chromatograph to a stable isotope ratio mass spectrometer has been developed and tested. The interface is usable for (13)C/(12)C determination of organic compounds, allowing measurement of small changes in (13)C abundance in individual analyte species. All of the carbon in each analyte is quantitatively converted into CO(2) while the analyte is still dissolved in the aqueous liquid phase. This is accomplished by an oxidizing agent such as ammonium peroxodisulfate. The CO(2) is separated from the liquid phase and transferred to the mass spectrometer. It is shown that the whole integrated process does not introduce isotope fractionation. The measured carbon isotope ratios are accurate and reproducible. The sensitivity of the complete system allows isotope ratio determination down to 400 ng of compound on-column. By-passing the high-performance liquid chromatography (HPLC) separation allows bulk isotopic analysis with substantially lower sample amounts than those required by conventional elemental analyzers. The results of the first applications to amino acids, carbohydrates, and drugs, eluted from various types of HPLC columns, are presented. The wide range of chromatographic methods enables the analysis of compounds never before amenable to isotope ratio mass spectrometry techniques and may lead to the development of many new assays.     

Formation of phosphopeptide-metal ion complexes in liquid chromatography/electrospray mass spectrometry and their influence on phosphopeptide detection

Despite major advances in mass spectrometry, the detection of phosphopeptides by liquid chromatography with electrospray mass spectrometry (LC/ES-MS) still remains very challenging in proteomics analysis. Phosphopeptides do not protonate efficiently due to the presence of one or more acidic phosphate groups, making their detection difficult. However, other mechanisms also contribute to the difficulties in phosphopeptide analysis by LC/ES-MS. We report here on one such undocumented problem: the formation of phosphopeptide-metal ion complexes during LC/ES-MS. It is demonstrated that both synthetic phosphopeptides and phosphopeptides from bovine beta-casein and alpha-casein form phosphopeptide-metal ion complexes containing iron and aluminum ions, resulting in a dramatic decrease in signal intensity of the protonated phosphopeptides. The interaction of phosphopeptides with metal ions on the surface of the C18 stationary phase is also shown to alter their chromatographic behavior on reversed-phase columns such that the phosphopeptides, especially multiply phosphorylated peptides, become strongly retained and very difficult to elute. The sources of iron and aluminum are from the solvents, stainless steel, glassware and C18 material. It was also found that, upon addition of EDTA, the formation of the phosphopeptide-metal ion complex is diminished, and the phosphopeptides that did not elute from the LC column can now be detected efficiently as protonated molecules. The sensitivity of detection was greatly increased such that a tetra-phosphorylated peptide, RELEELNVPGEIVEpSLpSpSpSEESITR from the tryptic digestion of bovine beta-casein, was detected at a limit of detection of 25 fmol, which is 400 times lower than without EDTA.     

Quantitation in gradient high performance liquid chromatography/inductively coupled mass spectrometry investigated using diclofenac and chlorpromazine

The use of directly coupled high performance liquid chromatography/inductively coupled plasma mass spectroscopy (HPLC/ICPMS) employing chlorine ((35)Cl/(37)Cl) detection has been investigated with respect to the detection and quantitation of the drugs diclofenac and chlorpromazine. By integration of peak areas in the 'chloratogram' (the chlorine specific HPLC chromatogram), a calibration curve was constructed, from which the concentrations could be determined. Chlorine detected HPLC/ICPMS is quantitative over a wide range of concentrations of pharmaceutical relevance for metabolite detection and the results reproducible (standard deviation +/- 0.43%) over multiple injections. Application of gradient chromatography and variation in the bulk mobile phase physicochemical properties has little effect on the ICPMS detection response for these compounds. This work indicates that the use of HPLC/ICPMS is likely to be quantitatively reliable for metabolism studies for a range of chlorinated xenobiotics.     

Selective detection of phosphopeptides in complex mixtures by electrospray liquid chromatography/mass spectrometry

A mass spectrometry-based method that does not involve the use of radiolabeling was developed for selective detection of phosphopeptides in complex mixtures. Mixtures of phosphorylated and nonphosphorylated peptides at the low picomole level are analyzed by negative ion electrospray liquid chromatography/mass spectrometry using C-18 packed fused-silica columns (≤320-μm i.d.). Peptides and phosphopeptides in the chromatographic eluant undergo collision-induced dissociation in the free-jet expansion region prior to the mass analyzing quadrupole. Using relatively high collisional excitation potentials, phospho|peptides containing phosphoserine, phosphothreonine, and phosphotyrosine fragment to yield diagnostic ions at m/z 63 and 79 corresponding to PO₂ ^?; and PO₃ ^?, respectively. Chromatographic peaks containing phosphopeptides are indicated where these diagnostic ions maximize. The highest sensitivity for phosphopeptide detection is obtained using selected-ion monitoring for m/z 63 and 79. Full-scan mass spectra that exhibit the diagnostic phosphopeptide fragment ions, together with pseudomolecular ions, may be obtained by stepping the collisional excitation potential from a high value during the portion of each scan in which the low-mass-to-charge ratio diagnostic marker ions are being detected to a lower value while the upper mass-to-charge ratio range is being scanned. Good sensitivity for phosphopeptide detection was achieved using standard trifluoroacetic acid containing mobile phases for reversed-phase high-performance liquid chromatography. Data illustrating the selectivity and sensitivity of the approach are presented for mixtures of peptides and phosphopeptides containing the three commonly phosphorylated amino acids.     

An online coupled peritoneal macrophage/cell membrane chromatography and high‐performance liquid chromatography/mass spectrometry method to screen for anti‐inflammatory components from the Chinese traditional medicine Chloranthus multistachys Pei

Cell membrane chromatography (CMC) is a chromatographic biological affinity method that uses specific cell membranes as the stationary phase. In this study, a novel peritoneal macrophage/cell membrane chromatography (PM/CMC)–online‐high performance liquid chromatography/mass spectrometry (HPLC/MS) method was established to screen for the anti‐inflammatory components from traditional Chinese medicines using hydrocortisone and dexamethasone as standards. The stationary phase of the CMC employed mouse peritoneal macrophage cell membranes. This method was applied to the purification and identification of components in extracts of Chloranthus multistachys Pei. The major component retained by CMC was identified as isofraxidin by HPLC/MS. In vitro experiments revealed that IF was able to inhibit the production of nitric oxide and tumor necrosis factor‐α in lipopolysaccharide‐stimulated mice and peritoneal macrophages in a dose‐dependent manner. The results demonstrated that the PM/CMC‐online‐HPLC/MS is an effective screening system for the rapid detection, enrichment, and identification of target components from complex samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass spectrometry.

A method for the determination of phosphorylation sites in phosphoproteins based on column-switching high-performance liquid chromatography (HPLC) has been developed. The HPLC system consisted of a titania precolumn for the selective adsorption of phosphopeptides, an anion-exchange analytical column and a UV detector (215 nm). Rabbit muscle phosphorylase a (RPa) and porcine stomach pepsin (PSP) were tested as model phosphoproteins. After protease digestion, the resulting phosphopeptides were successfully isolated by column-switching HPLC. The phosphopeptide fractions were analyzed by electrospray ionization mass spectrometry with a positive or negative ion mode after purification by reversed-phase HPLC. Pseudo-molecular ion peaks corresponding to Gln-Ile-Ser(p)-Val-Arg (MW 681.7) and Glu-Ala-Thr-Ser(p)-Gln-Glu-Leu (MW 856.8) were detected from the tryptic digest of RPa and chymotryptic digest of PSP, respectively, which agreed with the theoretically expected phosphopeptide fragments.     

Sequential Fe3O4/TiO2 enrichment for phosphopeptide analysis by liquid chromatography/tandem mass spectrometry

Protein phosphorylation regulates a wide range of cellular functions and is associated with signaling pathways in cells. Various strategies for enrichment of phosphoproteins or phosphopeptides have been developed. Here, we developed a novel sequential phosphopeptide enrichment method, using magnetic iron oxide (Fe₃O₄) and titanium dioxide (TiO₂) particles, to detect mono‐ and multi‐phosphorylated peptides. In the first step, phosphopeptides were captured on Fe₃O₄ particles. In a subsequent step, any residual phosphopeptides were captured on TiO₂ particles. The particles were eluted and rinsed to yield phosphopeptide‐enriched fractions that were combined and analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The validity of this sequential Fe₃O₄/TiO₂ enrichment strategy was demonstrated by the successful enrichment of bovine α‐casein phosphopeptides. We then applied the sequential Fe₃O₄/TiO₂ enrichment method to the analysis of phosphopeptides in L6 muscle cell lysates and successfully identified mono‐ and multi‐phosphorylated peptides. Copyright © 2010 John Wiley & Sons, Ltd.     

Dynamic identification of phosphopeptides using immobilized metal ion affinity chromatography enrichment, subsequent partial beta-elimination/chemical tagging and matrix-assisted laser desorption/ionization mass spectrometric analysis

The enrichment of phosphopeptides using immobilized metal ion affinity chromatography (IMAC) and subsequent mass spectrometric analysis is a powerful protocol for detecting phosphopeptides and analyzing their phosphorylation state. However, nonspecific binding peptides, such as acidic, nonphosphorylated peptides, often coelute and make analyses of mass spectra difficult. This study used a partial chemical tagging reaction of a phosphopeptide mixture, enriched by IMAC and contaminated with nonspecific binding peptides, following a modified beta-elimination/Michael addition method, and dynamic mass analysis of the resulting peptide pool. Mercaptoethanol was used as a chemical tag and nitrilotriacetic acid (NTA) immobilized on Sepharose beads was used for IMAC enrichment. The time-dependent dynamic mass analysis of the partially tagged reaction mixture detected intact phosphopeptides and their mercaptoethanol-tagged derivatives simultaneously by their mass difference (-20 Da for each phosphorylation site). The number of new peaks appearing with the mass shift gave the number of multiply phosphorylated sites in a phosphopeptide. Therefore, this partial chemical tagging/dynamic mass analysis method can be a powerful tool for rapid and efficient phosphopeptide identification and analysis of the phosphorylation state concurrently using only MS analysis data.     

Analysis of thiamine in dried yeast by high-performance liquid chromatography and high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry

Summary High-performance liquid chromatography (HPLC) and high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (HPLC/APCI-MS) have been applied to the analysis of thiamine in dried yeast. Thiamine was extracted from dried yeast with isobutanol containing sodium 1-octanesulfonate as an ion-pairing agent and determined by HPLC on a reversed phase ODS column with UV detection at 254 nm. Response was linear in the range 25–300 μg/g of thiamine in dried yeast with a coefficient of variation in the reproducibility of 8.0%. Thiamine was recovered in good yield (109.2%, n=5). Identification of the thiamine peak was obtained by the mass spectrum using the HPLC/APCI-MS system. The utility of the selected ion monitoring technique using the HPLC/APCI-MS was also investigated. The results obtained by this method are in good agreement with those obtained by the thiochrome method [1].     

Chemo-affinity of titania for the column-switching HPLC analysis of phosphopeptides.

A column-switching high-performance liquid chromatography (HPLC) system for the determination of phosphopeptides has been developed. The method is based on the selective adsorption of phosphopeptides on a titania (TiO2) precolumn and successive HPLC separation of the phosphopeptides on an anion-exchange column with a UV detector (215 nm). The recoveries of phosphopeptides were tested using authentic phosphopeptides [Gln-Ile-Ser(p)-Val-Arg, Ile-Ser(p)-Val-Arg and Lys-Gln-Ile-Ser(p)-Val-Arg] at an injection amount of 1 microg. The recoveries were 74.3, 79.6, and 82.6%, respectively, while the corresponding dephosphopeptides were not retained on the titania precolumn.     

Phosphopeptide analysis by directly coupling two-dimensional planar electrochromatography/thin-layer chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A novel strategy is presented for the fractionation of complex peptide mixtures using two-dimensional planar electrochromatography/thin-layer chromatography (2D PEC/TLC). Phosphopeptides migrate more slowly in the first dimension, based upon their anionic phosphate residues, and certain predominantly acidic phosphopeptides even migrate in the opposite direction, relative to the bulk of the peptides. Phosphopeptides are further distinguished based upon hydrophilicity in the second dimension. This permits a restricted region of the plate to be directly interrogated for the presence of phosphopeptides by mass spectrometry (MS). Phosphopeptide analysis from the plates by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-MS and tandem MS enabled peptide sequencing and identification.     

Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry

On-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization-mass spectrometry (IMAC/CE/ESI-MS) offers selective preconcentration of phosphorylated peptides with identification of the phosphorylated amino acid(s). The preconcentration provides low concentration limits of detection and capillary electrophoresis separates the peptides. Recently, we reported a fast, simple, and sensitive on-line IMAC/CE/ESI-MS/MS method for the determination of phosphopeptides at low-pmole levels. That work is expanded here by use of multiple stage tandem mass spectrometry (MS(n), n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI-MS(n) is demonstrated by the analysis of tryptic digests of alpha- and beta-casein and in-gel tryptic digests of beta-casein.     

Arsenosugar identification in seaweed extracts using high-performance liquid chromatography/electrospray ion trap mass spectrometry

The development of analytical techniques suitable for providing structural information on a wide range of elemental species is a growing necessity. For arsenic speciation a variety of mass spectrometric techniques, mainly inductively coupled plasma mass spectrometry (ICP-MS) and electrospray tandem mass spectrometry (ES-MS/MS) coupled on-line with high-performance liquid chromatography (HPLC), are in use. In this paper we report the identification of arsenic species present in samples of marine origin (seaweed extracts) using ES ion trap mass spectrometry (IT) multistage mass spectrometry (MS(n)). Both reversed-phase and anion-exchange HPLC have been coupled on-line to ES-ITMS. Product ion scans with multiple stages of tandem MS (MS(n); n=2-4) were used to acquire diagnostic data for each arsenosugar. The spectra contain structurally characteristic fragment ions for each of the arsenosugars examined. In addition it was observed that upon successive stages of collision-induced dissociation (CID) a common product ion (m/z 237) was formed from all four arsenosugars examined. This product ion has the potential to be used as an indicator for the presence of dimethylated arsenosugars (dimethylarsinoylribosides). The HPLC/ES-ITMS(n) method developed allows the sensitive identification of arsenosugars present in crude seaweed extracts without the need for extended sample preparation. In fact, sample preparation requirements are identical to those typically employed for HPLC/ICP-MS analysis. Additionally, the resulting product ions are structurally diagnostic of the arsenosugars examined, and tandem mass spectra are reproducible and correspond well to those obtained using other low-energy CID techniques. As a result, the HPLC/ES-ITMS(n) approach minimises the potential for arsenic species misidentification and has great potential as a means of overcoming the need for characterised standards.     

Zirconia-based column high-performance liquid chromatography/atmospheric pressure photoionization tandem mass spectrometric analyses of drug molecules in rat plasma

A method using zirconia-based column high-performance liquid chromatography (HPLC) interfaced with an atmospheric pressure photoionization (APPI) source and a tandem mass spectrometer (MS/MS) was developed for the quantitative determination of new chemical entities in rat plasma in support of pharmacokinetics studies. The ionization suppression resulting from endogenous components of the biological matrices on the quantitative zirconia-based column HPLC/APPI-MS/MS method was investigated using the post-column infusion technique. The analytical results for 'rapid rat pharmacokinetics' for 12 drug discovery compounds, obtained by both silica-based phase (S-phase) and zirconia-based phase (Z-phase) chromatographic separation, are in good agreement in terms of accuracy. The application of a Z-phase column for high-temperature fast HPLC/MS/MS methods was explored to reduce the analysis time from 3 min to 30 s for column temperatures of 25-110 degrees C, respectively. The chromatographic retention times and peak responses of all analytes were found to be reproducible under high-temperature conditions following 100 continuous injections, with %CV less than 0.4 and 5, respectively.     

Interfacing liquid chromatography with atmospheric pressure MALDI-MS

Two different strategies for coupling liquid chromatography with atmospheric pressure matrix assisted laser desorption/ionization (AP MALDI) are presented. The first method is flow-injection liquid AP UV-MALDI. Compared with previous similar research, the detection limit was improved 10 times to 8.3 fmol using a solution of 50 nM peptide with 25 mM α-cyano-4-hydroxycinnamic acid. The applicability of this method to measure oligosaccharides, actinomycin antibiotics, antibiotics, phosphopeptides, and proteins is demonstrated. The upper mass limit achieved with the current instrumentation is 6,500 Da (doubly charged cytochrome c). The feasibility of a second strategy based on single-droplet IR AP MALDI is demonstrated here. Aqueous peptide solutions were successfully measured by this method.     

Investigation of analytical variation in metabonomic analysis using liquid chromatography/mass spectrometry

Sources of analytical variation in high-performance liquid chromatography/mass spectrometry (HPLC/MS), such as changes in retention, mass accuracy or signal intensity, have been investigated to assess their importance as a variable in the metabonomic analysis of human urine. In this study chromatographic retention and mass accuracy were found to be quite reproducible with the most significant source of analytical variation in the data sets obtained being the result of changes in detector response. Depending on the signal intensity threshold used to define the presence of a peak a sample component could be present in some replicate injections and absent in others within the same run. The implementation of a more sophisticated data software analysis package was found to greatly reduce the impact of detector response variability resulting in improved data analysis.     

Description and validation of a staggered parallel high performance liquid chromatography system for good laboratory practice level quantitative analysis by liquid chromatography/tandem mass spectrometry.

The Aria LX4 staggered parallel high performance liquid chromatography (HPLC) system is evaluated for application to good laboratory practice (GLP) level quantitative analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS). This system consists of four fully independent binary HPLC pumps, a modified autosampler, and a series of switching and selector valves all controlled by a single computer program. The system improves sample throughput without sacrificing chromatographic separation or data quality. Validation results for four different compounds, each analyzed on a separate channel of the Aria system, show precision and accuracy equivalent to that required of a single-channel system. The results show that sample throughput can be increased nearly four-fold without requiring significant changes in current analytical procedures. The flexibility and ease of use of the Aria system suggest that it should be possible to quickly implement it in any analytical LC/MS/MS environment.     

Application of on-line nano-liquid chromatography/mass spectrometry in metabolite identification studies

Metabolite identification is an important part of the drug discovery and development process. High sensitivity is necessary to identify metabolic products in vitro and in vivo. The most common method utilizes standard high-performance liquid chromatography (4.6 mm i.d. column and 1 mL/min flow rate) coupled to tandem mass spectrometry (HPLC/MS/MS). We have developed a method that utilizes a nano-LC system coupled to a high-resolution tandem mass spectrometer to identify metabolites from in vitro and in vivo samples. Using this approach, we were able to increase the sensitivity of analysis by approximately 1000-fold over HPLC/MS. In vitro samples were analyzed after simple acetonitrile precipitation, centrifugation, and dilution. The significant improvement in sensitivity enabled us to conduct experiments at very low substrate concentrations (0.01 μM), and very low incubation volumes (20 μL). In vivo samples were injected after simple dilution without any pre-purification. All the metabolites identified by conventional HPLC/MS/MS were also identified using the nano-LC method. This study demonstrates a very sensitive approach to identifying phase I and II metabolites with throughput and separation equivalent to the standard HPLC/MS/MS method.     

Porous graphitic carbon chromatography/tandem mass spectrometric determination of cytarabine in mouse plasma

A high-performance liquid chromatography (HPLC) system using a porous graphitic carbon (PGC) stationary phase interfaced with an electrospray ionization (ESI) source and a tandem mass spectrometer (MS/MS) for the analysis of cytarabine (ara-C) in mouse plasma samples has been developed in support of a pharmacodynamic study. The graphitized carbon column was adopted for the separation of ara-C and endogenous peaks from mouse plasma samples under the reversed-phase phase mode in liquid chromatography. The retention characteristics of the PGC column and the ionization efficiencies of all analytes based on the experimental factors such as the composition of mobile phases were investigated. The potential of ionization suppression resulting from the endogenous biological matrices on the PGC column during HPLC/ESI-MS/MS was investigated using post-column infusion. The concentrations of ara-C in mouse plasma obtained by using PGC-HPLC/MS/MS and ion-pairing HPLC/MS/MS were found to be in good agreement in terms of analytical accuracy.