Possibilities of multivariate curve resolution and partial least squares in the resolution of coeluted peaks in liquid chromatography with electrochemical detection

Two different strategies are compared for the resolution of coeluted peaks in liquid chromatography with electrochemical detection. The first is voltammetric detection (VD) for the acquisition of currents as a function of potential and time with multivariate curve resolution by alternating least squares (MCR-ALS) for data analysis and quantification. The second is amperometric detection (AD), i.e. recording currents as a function of time for a fixed potential and calibration by partial least squares (PLS). Both approaches are used to analyse a model mixture of pyrocatechol, dopamine and epinephrine. The results show that for high analyte concentrations VD-MCR-ALS provides accurate quantification with minimal effort (a single injection of the sample and each standard). However, at lower concentrations the excessive proportion of noise and the predominance of the capacitive contribution decrease the performance of VD-MCR-ALS, thus making AD-PLS preferable as its greater accuracy counters the more time-consuming calibration, which involves the injection of a large number of mixtures of different composition.     

Ligand-exchange detection of phosphorylated peptides using liquid chromatography electrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is used to selectively detect analytes with a high affinity for metal ions. The detection method is based on the selective monitoring of a competing ligand at its specific m/z value that is released during the ligand-exchange reaction of a metal-ligand complex with analyte(s) eluting from a reversed-phase liquid chromatography column. The ligand-exchange reaction proceeds in a postcolumn reaction detection system placed prior to the inlet of the electrospray MS interface. The feasibility of metal affinity detection by ESI-MS is demonstrated using phosphorylated peptides and iron(III)methylcalcein blue as reactant, as a model system. Methylcalcein blue (MCB) released upon interaction with phosphorylated peptides is detected at m/z 278. The ligand-exchange detection is coupled to a C8 reversed-phase column to separate several nonphosphorylated enkephalins and the phosphorylated peptides pp60 c-src (P) and M2170. Detection limits of 2 microM were obtained for pp60 c-src (P) and M2170. The linearity of the detection method is tested in the range of 2-80 micromol/L phosphorylated compounds (r(2) = 0.9996), and a relative standard deviation of less than 8% (n = 3) for all MCB responses of the different concentrations of phosphorylated compounds was obtained. The presented method showed specificity for phosphorylated peptides and may prove a useful tool for studying other ligand-exchange reactions and metal-protein interactions.     

High-performance liquid chromatography of lignin-derived phenols in environmental samples with diode array detection

Lignin-derived and standard phenols were successfully analyzed with reversed-phase high-performance liquid chromatography (HPLC) with diode array detection. The 11 major phenols produced by cupric oxide (CuO) oxidation were clearly and rapidly separated. Determination by diode array detection also allowed detection of interfering impurities within individual HPLC peaks. The lignin phenols were accordingly corrected and quantified. This method yields precise and reliable data for various environmental samples such as dissolved organic matter from aqueous samples and sediments and can also be applied to various particulate materials such as detritus and plant tissues. An interlaboratory method comparison with humic substance standards from the International Humic Substances Society revealed differences in lignin phenol concentrations, whereas lignin parameters better coincided.     

Background correction and multivariate curve resolution of online liquid chromatography with infrared spectrometric detection

The use of multivariate curve resolution-alternating least-squares (MCR-ALS) in liquid chromatography-infrared detection (LC-IR) is troublesome due to the intense background absorption changes during gradient elution. Its use has been facilitated by previous removal of a significant part of the solvent background IR contributions due to common mobile phase systems employed during reversed phase gradient applications. Two straightforward background correction approaches based on simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) and principal component analysis (PCA) are proposed and evaluated on reversed phase gradient LC-IR data sets obtained during the analysis of carbohydrate and nitrophenol mixtures. After subtraction of the calculated background signal, MCR-ALS provided improved signal-to-noise ratios, removed remaining mobile phase and background signal contributions, and resolved overlapping chromatographic peaks. The present approach tends to enable easy-to-use background correction to facilitate the use of MCR-ALS in online LC-IR, even in challenging situations when gradient conditions are employed and only poor chromatographic resolution is achieved. It, therefore, shows great potential to facilitate the full exploitation of the advantages of simultaneous quantification and identification of a vast amount of analytes employing online IR detection, making new exciting applications more accessible.     

Separation and detection of siderophores produced by marine bacterioplankton using high-performance liquid chromatography with electrospray ionization mass spectrometry

High-performance liquid chromatography-electrospray ionization mass spectrometry was applied to the detection of the iron(III) complexes of the hydroxamate siderophores rhodotoluric acid, deferrioxamine B, and deferrichrome. Separation of the iron(III) complexes was obtained using a polystyrene-divinylbenzene stationary phase. The retention and responses of ferrioxamine and ferrichrome were optimal when a gradient elution program with methanol and 0.1% (v/v) formic acid as the mobile phases was used. These conditions were also suitable for the retention and separation of the uncomplexed ligands. Retention of iron(III) rhodotoluate was improved when formic acid was replaced by the ion-pairing reagent heptafluorobutyric acid (0.1%). Detection limits for the ferric complexes, defined as 3 SD of the lowest determined standard, were 26 nM for iron(III) rhodotoluate, 0.23 nM for ferrioxamine, and 0.40 nM for ferrichrome. A protocol for the solid-phase extraction of these hydroxamate siderophores from seawater was developed and applied to the extraction of siderophores from enriched incubated seawater samples.     

Combining liquid chromatography with MALDI mass spectrometry using a heated droplet interface

A novel interfacing technology is described to combine solution-based separation techniques such as liquid chromatography (LC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The interface includes a transfer tube having an inlet and an outlet, the inlet being adapted to accept the LC effluents and the outlet being adapted to form continuously replaced, hanging droplets of the liquid stream, and a MALDI sample plate mounted below the outlet of the transfer tube for collecting the droplets. The liquid stream in the transfer tube is heated to a temperature sufficient to cause partial evaporation of the carrier solvent from the hanging droplets. The droplets are dislodged to the MALDI plate, which is heated to above the boiling point of the carrier solvent to cause further evaporation of the carrier solvent from the collected droplets. It is found that analytes can be fractionated and deposited to a sample spot of 0.8 mm in diameter when a liquid flow rate of up to 50 microL/min and a fractionation interval of 1 min/spot are used. Flow rate of up to 200 microL/min can be used with a deposition sample spot of 2.4 mm in diameter on a commercial MALDI target. This heated droplet interface does not introduce sample loss, and the detection sensitivity of LC/MALDI is similar to that of standard MALDI, i.e., low femtomoles for peptide analysis with a microliter sample deposition. It is compatible with microbore and narrow-bore column separation, thus allowing the injection of a larger amount of sample for separation and analysis, compared to a capillary column LC/MALDI system. The detection dynamic range is shown to be in the order of 10(6) for peptide mixture analysis, which is 4 orders of magnitude greater than standard MALDI. The application of this interface for combining LC with MALDI MS/MS is demonstrated in the proteome analysis of water-soluable protein components of E. coli K12 extracts.     

Investigation of the immunogenicity of a protein drug using equilibrium dialysis and liquid chromatography tandem mass spectrometry detection

Biotherapeutics such as protein and peptide drugs have attracted significant attention in the medical community and pharmaceutical industry in recent years. Immunogenicity is one of the major concerns in the development and application of biotherapeutics. Although great efforts have been put forth in reducing immunogenicity, monitoring the free ("active") drug concentration and the antibody formation is critical for preclinical and clinical studies. Currently, it is still a challenging task to have a standardized test method monitoring immunogenicity when biotherapeutic compounds such as proteins and peptides are administrated. Combined with liquid chromatography/tandem mass spectrometry detection, the equilibrium dialysis technique that is conventionally used for measuring the free and bound concentration of small organic molecules was extended to the application of measuring the free and bound concentrations of a protein drug with a relative molecular mass over 10,000 from plasma samples containing antibody. This novel approach could also be used for accurately measuring the antibody concentration when a reference standard of the antibody is available.     

Analysis of protein phosphorylation by capillary liquid chromatography coupled to element mass spectrometry with 31P detection and to electrospray mass spectrometry

A method for phosphopeptide identification by capillary liquid chromatography (muLC) interfaced alternatively to element mass spectrometry (inductively coupled plasma mass spectrometry, ICPMS) and to electrospray ionization mass spectrometry (ESI-MS) is described. ICPMS is used for 31P detection and ESI-MS provides the corresponding molecular weight information. Alignment of the two separate muLC runs is performed using the baseline distortion at the elution front, which shows up in both muLC-ICPMS and muLC-ESI-MS. Both a quadrupole and a magnetic sector field mass analyzer were used in combination with ICP. The detection limit achieved for the muLC-ICP-HRMS runs is approximately 0.1 pmol of phosphopeptide injected. Without any further precautions, contamination by phosphate-containing compounds at this level was found to be uncritical. The method is demonstrated for the analysis of a complex mixture of synthetic phosphopeptides and a set of tryptic digests of three phosphoproteins. These include beta-casein, activated human MAP kinase ERK1, and protein kinase A catalytic subunit. The tryptic phosphopeptides of these proteins could all be detected and identified by our new strategy. Analysis of three fractions of protein kinase A catalytic subunit with different phosphorylation status gives direct access to the order in which the phosphorylation of the four phosphorylation sites occurs. The two most important aspects of using muLC-ICPMS with 31P detection for phosphopeptide identification are (i) that a high selectivity is achieved and (ii) that the signal intensity is independent of the chemical form of phosphorus and directly proportional to the molar amount of 31P in the muLC eluate. Thus, muLC-ICPMS with 31P detection is introduced as a new, robust, and specific method in phosphoproteomics.     

High-throughput screening for enzyme inhibitors using frontal affinity chromatography with liquid chromatography and mass spectrometry

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated Kd value of 0.3 microM. Other binders yielded Kd values ranging from 0.35 to 3.35 microM. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.     

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.     

Study of complexation in methanol/water mixtures by infrared and Raman spectroscopy and multivariate curve resolution-alternating least-squares analysis

The structure of the mobile phase in liquid chromatography plays an important role in the determination of retention behavior on reversed-phase stationary materials. One of the most commonly employed mobile phases is a mixture of methanol and water. In this work, infrared and Raman spectroscopic methods were used to investigate the structure of species formed in methanol/water mixtures. Chemometric methods using multivariate curve resolution by alternating least-squares analysis were used to resolve the overlapped spectra and to determine concentration profiles as a function of composition. The results showed that the structure of these mixtures could be described by a mixture model consisting of four species, namely, methanol, water, and two complexes, methanol/water (1:1) and methanol/water (1:4). The spectral frequencies and concentration profiles found from the Raman and infrared measurements were consistent with one another and with theoretical calculations.     

Sulfur-specific detection of impurities in cimetidine drug substance using liquid chromatography coupled to high resolution inductively coupled plasma mass spectrometry and electrospray mass spectrometry

The use of liquid chromatography coupled to sector field inductively coupled plasma mass spectrometry (SF-ICP-MS) for the specific detection of sulfur-containing compounds is described. In the sulfur-containing drug substance cimetidine, structurally related impurities well below the 0.1% mass fraction level relative to the main drug substance could easily be detected. The structure of most of the impurities was confirmed by electrospray mass spectrometry (ESI-MS), and thus, the complementarity of the two techniques for drug analysis is shown. The limit of detection by SF-ICP-MS for cimetidine in solution was approximately 4-20 ng x g(-1), but it was blank-limited.     

Nanoscale packed-capillary liquid chromatography coupled with mass spectrometry using a coaxial continuous-flow fast atom bombardment interface

Nanoscale packed-capillary liquid chromatography (LC) columns have been coupled with mass spectrometry (MS) using a coaxial continuous-flow fast atom bombardment interface. The combined system has been applied to the analysis of mixtures of peptides, including synthetic mixtures of bioactive peptides and tryptic digests of proteins. Nanoscale packed-capillary columns offer two principal advantages for LC/MS analysis--high chromatographic separation efficiencies and low mobile-phase flow rates. The high separation efficiencies facilitate the separation of complex mixtures, and the low mobile-phase flow rates reduce problems with coupling the LC effluent with the high-vacuum, high-voltage environment of sector MS ion sources. The columns used in this work were 50- or 75-micron i.d., 1-2 m long, packed with 10-micron C18 particles, using mobile-phase flow rates of 50-350 nL/min.     

Development and evaluation of a candidate reference method for the determination of total cortisol in human serum using isotope dilution liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry

Cortisol is an important diagnostic marker for the production of steroid hormones, and accurate measurements of serum cortisol are necessary for proper diagnosis of adrenal function. A candidate reference method involving isotope dilution coupled with liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. An isotopically labeled internal standard, cortisol-d(3), was added to serum, followed by equilibration and solid-phase and ethyl acetate extractions to prepare samples for liquid chromatography/mass spectrometry electrospray ionization (LC/MS-ESI) and liquid chromatography/tandem mass spectrometry electrospray ionization (LC/MS/MS-ESI) analyses. (M + H)(+) ions at m/z 363 and 366 for cortisol and its labeled internal standard were monitored for LC/MS. The transitions of (M + H)(+) --> [(M + H)(+) - 2H(2)O] at m/z 363 --> 327 and 366 --> 330 were monitored for LC/MS/MS. The accuracy of the measurement was evaluated by a comparison of results of this candidate reference method on lyophilized human serum reference materials for cortisol [Certified Reference Materials 192 and 193] with the certified values determined by gas chromatography/mass spectrometry reference methods and by a recovery study for the added cortisol. The results of this method for total cortisol agreed with the certified values within 1.1%. The recovery of the added cortisol ranged from 99.8% to 101.0%. This method was applied to the determination of cortisol in samples of frozen serum pools. Excellent precision was obtained with within-set CVs of 0.3%-1.5% and between-set CVs of 0.04%-0.4% for both LC/MS and LC/MS/MS analyses. The correlation coefficients of all linear regression lines ranged from 0.998 to 1.000. The detection limits (at a signal-to-noise ratio of approximately 3-5) were 10 and 15 pg for LC/MS and LC/MS/MS, respectively. This method, which demonstrates good accuracy and precision, and is free from interferences from structural analogues, qualifies as a candidate reference method and can be used as an alternative reference method to provide an accuracy base to which the routine methods can be compared.     

Simultaneous detection and identification of O-GlcNAc-modified glycoproteins using liquid chromatography-tandem mass spectrometry

Glycoproteins carrying O-linked N-acetylglucosamine (O-GlcNAc) modifications have been isolated from a wide range of organisms ranging from trypanosomes to humans. Interest in this modification is increasing as evidence accumulates that it is an abundant and transient modification that is dynamic and responsive to cellular stimuli. Concurrent advances in biological mass spectrometry (MS) have facilitated high-sensitivity protein identification by tandem MS. In this study, we show that the lability of the O-GlcNAc moiety to low-energy collision in tandem MS offers a means of distinguishing such peptides from others that are not modified. The differential between the energy required to remove the O-GlcNAc group and the energy required to fragment the peptide chain allows the O-GlcNAc group to be detected and the peptide sequence, and therefore the protein, to be identified. This technique thus allows the simultaneous detection and identification of O-GlcNAc-modified peptides, even when present at low levels in complex mixtures. The method was initially developed and validated using a synthetic O-GlcNAc-modified peptide and then applied to the detection of an extremely low abundance O-GlcNAc-modified peptide from bovine alpha-crystallin. We believe that with further development this assay system may prove to be a useful tool for the direct investigation of intracellular O-GlcNAc levels, thus providing valuable insights into the physiological role of O-GlcNAc modified proteins.     

Urine testing for designer steroids by liquid chromatography with androgen bioassay detection and electrospray quadrupole time-of-flight mass spectrometry identification

New anabolic steroids show up occasionally in sports doping and in veterinary control. The discovery of these designer steroids is facilitated by findings of illicit preparations, thus allowing bioactivity testing, structure elucidation using NMR and mass spectrometry, and final incorporation in urine testing. However, as long as these preparations remain undiscovered, new designer steroids are not screened for in routine sports doping or veterinary control urine tests since the established GC/MS and LC/MS/MS methods are set up for the monitoring of a few selected ions or MS/MS transitions of known substances only. In this study, the feasibility of androgen bioactivity testing and mass spectrometric identification is being investigated for trace analysis of designer steroids in urine. Following enzymatic deconjugation and a generic solid-phase extraction, the samples are analyzed by gradient LC with effluent splitting toward two identical 96-well fraction collectors. One well plate is used for androgen bioactivity detection using a novel robust yeast reporter gene bioassay yielding a biogram featuring a 20-s time resolution. The bioactive wells direct the identification efforts to the corresponding well numbers in the duplicate plate. These are subjected to high-resolution LC using a short column packed with 1.7-microm C18 material and coupled with electrospray quadrupole time-of-flight mass spectrometry (LC/QTOFMS) with accurate mass measurement. Element compositions are calculated and used to interrogate electronic substance databases. The feasibility of this approach for doping control is demonstrated via the screening of human urine samples spiked with the designer anabolic steroid tetrahydrogestrinone. Application of the proposed methodology, complementary to the established targeted urine screening for known anabolics, will increase the chance of finding unknown emerging designer steroids, rather then being solely dependent on findings of the illicit preparations themselves.     

Recognition of trypsin autolysis products by high-performance liquid chromatography and mass spectrometry

Potential artifactual contributions are assessed in high-pressure liquid chromatograms and fast atom bombardment mass spectra from autolysis of different preparations of the widely used protease trypsin. Both commercially supplied and laboratory-purified samples were examined. Bovine pancreatic trypsin (1 mg/mL) was found to be completely destroyed in 2 h at pH 8.5, degraded to a complex mixture of small peptides which were characterized by their molecular weights. Some identifications were supported by sequencing by tandem mass spectrometry or by mass spectrometric analysis of the mixture resulting from a single Edman degradation. Autolysis of porcine pancreatic trypsin produced a completely different set of peptides. Five sites of hydrolysis at asparagine residues in bovine trypsin were also identified.     

Initial characterization of humic acids using liquid chromatography at the critical condition followed by size-exclusion chromatography and electrospray ionization mass spectrometry

Structural information on humic acids is difficult to obtain because of the heterogeneity of the acids. Herein liquid chromatography at the critical condition, LCCC, is used to provide a sorting mechanism for the diverse types of molecules contained in humic acids. The critical condition of polymers that are believed to model some subunit of the humic acid is determined. Humic acids from three different terrestrial sources (soil, compost, and peat) are then separated under these chromatographic conditions. The portion of the humic acid that has structure similar to that of the model polymer elutes at the retention volume of the critical condition of the model. Next, fractions are collected and further characterized. This detailed characterization includes high-efficiency size-exclusion chromatography and electrospray mass spectrometry. The size-exclusion chromatograms of the fractions were found to be markedly different from that of the original humic acid sample. This is strong evidence that the LCCC separation mechanism is different from size fractionation. The mass spectra of the humic acid fractions were also markedly different from those of the bulk humic acids previously reported. The mass spectra of specific fractions collected had repeating clusters of m/z values, which is more evidence that the critical condition separation is a powerful sort function.     

Monitoring and modeling of protein processes using mass spectrometry, circular dichroism, and multivariate curve resolution methods

Mass spectrometry has recently become one of the major analytical tools to study biomolecular structure and function. Ionization techniques, such as electrospray ionization (ESI), desorb biomolecules from solution to the gas phase keeping practically intact their natural structure. ESI applied to a protein solution produces a mixture of multiply charged ions, the ion charge distribution of which depends on the oligomeric form (mass) and on the protein surface exposed (amount of accommodated charges) of the related protein conformation. ESI-MS provides an efficient way to monitor protein processes; however, the ionic contributions of the different protein conformations involved usually overlap, and the use of chemometric tools is necessary to unravel the information related to the pure conformations that the biomolecule adopts along the process. Multivariate curve resolution-alternating least squares applied to MS-monitored protein processes provides the concentration profiles associated with the different protein conformations occurring during the process and the related pure mass spectra. The concentration profiles, in this context, the ionic contributions, describe the process mechanism and the structural information derived from the pure mass spectra characterizes the involved conformations. Mass spectra can be expressed schematically through percentages of base peak intensity. This chemical transformation compresses significantly the raw spectra and allows for an easier application of natural MS-related constraints, such as the presence of only one maximum, i.e., the base peak of a particular conformation, into the resolution of the pure signals. The combination of mass spectrometry and multivariate curve resolution methods is used to elucidate the mechanism of the pH-induced conformation changes of the bovine beta-lactoglobulin. As a final step, MS data are fused with circular dichroism data and are simultaneously analyzed to ensure and confirm that all the previously detected MS conformations really exist in solution and are an artifact of neither the ionization process nor their chemometric resolution.     

Integration of continuous-flow accelerator mass spectrometry with chromatography and mass-selective detection

Physical combination of an accelerator mass spectrometry (AMS) instrument with a conventional gas chromatograph-mass spectrometer (GC/MS) is described. The resulting hybrid instrument (GC/MS/AMS) was used to monitor mass chromatograms and radiochromatograms simultaneously when (14)C-labeled compounds were injected into the gas chromatograph. Combination of the two instruments was achieved by splitting the column effluent and directing half to the mass spectrometer and half to a flow-through CuO reactor in line with the gas-accepting AMS ion source. The reactor converts compounds in the GC effluent to CO2 as required for function of the ion source. With cholesterol as test compound, the limits of quantitation were 175 pg and 0.00175 dpm injected. The accuracy achieved in analysis of five nonzero calibration standards and three quality control standards, using cholesterol-2,2,3,4,4,6-d6 as injection standard, was 100 +/- 11.8% with selected ion monitoring and 100 +/- 16% for radiochromatography. Respective values for interday precision were 1.0-3.2 and 22-32%. Application of GC/MS/AMS to a current topic of interest was demonstrated in a model metabolomic study in which cultured primary hepatocytes were given [(14)C]glucose and organic acids excreted into the culture medium were analyzed.