Identification of drug metabolites in biological matrices by intelligent automated liquid chromatography/tandem mass spectrometry

A rapid and systematic strategy for the identification of drug metabolites in biological matrices based on liquid chromatography-tandem mass spectrometry (LC/MS/MS) techniques was utilized for the identification of drug metabolites of the HIV protease inhibitor Indinavir. This strategy integrates intelligent realtime mass spectrometry with HPLC detection and a predictive strategy for detecting metabolites arising from common biotransformations, to rapidly elucidate structures of drug metabolites. Structures of metabolites generated from in vitro incubation mixtures of Indinavir were characterized from a single chromatographic analysis using the automated LC/MS/MS methodology, thus reducing data acquisition time and improving efficiency.     

Rapid assessment of early glycation products by mass spectrometry

In order to detect the early glycation products, we have reacted a model peptide (t-boc-lys-ala-ala) with L-threose (a degradation product of ascorbic acid) and analyzed the reaction products by a combination of HPLC and mass spectrometry. Amino group modification, as observed by a fluorescamine assay, indicated complete modification after 3 days of incubation with a 10-fold excess of threose. As much as 60% of the adducts were acid labile and only 4% of the adducts could be observed by amino acid analysis. However, Fast atom bombardment mass spectrometry (FABMS) of the samples incubated for 6 hr showed relative molecular masses consistent with the formation of adducts corresponding to the addition of one and two molecules of L-threose to the peptide. Likewise, samples incubated for 12 hr showed peptide adducts with two and three L-threoses. The number of threose molecules added to the peptide was also confirmed from the FABMS analysis by using [1-13C]-threose as the glycating agent.     

Studies on the selection of new matrices for ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A new group of compounds has been successfully tested as matrices for ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI MS). Several new matrices for UV-MALDI MS have been found by choosing, as potential matrices, compounds that perform an intramolecular proton transfer along an intramolecular H-bond under UV irradiation. Compounds of this type are, for example, salicylamide, salicylanilide, several ortho-hydroxyacetophenones and ortho-hydroxybenzophenones. The matrix activity of these compounds is compared to the corresponding meta- and para-isomers and to the matrix activity of such well known matrices as 2,5-dihydroxybenzoic acid and 2,4,6-trihydroxyacetophenone. It was found that meta- and para-substituted hydroxycarbonyl compounds show either a significantly lower or no matrix activity compared with the ortho isomers.     

Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides

Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4. Both sequence and linkage data are obtained for N-acetyllactosamine and sialyl-N-acetyllactosamine oligosaccharide antennae from biantennary glycans using MS3, and the location of a bisecting GlcNAc residue is also established after exposing the core pentasaccharide. Higher-order experiments further illustrate the potential of electrospray ionization-quadrupole ion trap mass spectrometry for carbohydrate analysis, as MS8 is used to produce significant and otherwise unobtainable branching information for an oligosaccharide from chicken ovalbumin. These studies constitute further evidence of the unique role that ion trap mass spectrometry can assume in the area of oligosaccharide analysis.     

Tandem mass spectrometry analysis of synthetic opioid peptide analogs

Five synthetic opioid peptides that were designed to have specific opioid receptor-binding properties were studied by low energy collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). The MS/MS data are required for the analysis of those peptides in ovine plasma in a study to determine the placental transfer of the peptide to the fetus. The synthetic enkephalin-related peptides were: Tyr-D-Arg-Phe-Lys-NH2, (DALDA), N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH, (ICI 174,864), Tyr-D-Thr-Gly-Phe-Leu-Thr, (DTLET), Tyr-D-Pen-Gly-Phe-D-Pen-OH, (DPDPE), and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2, (CTAP). Liquid secondary ion mass spectrometry (LSIMS) was used for sample desorption-ionization, and a hybrid (E1BE2qQ) tandem mass spectrometer was used to collect the product-ion spectra. A protonated molecule ion, [M + H]+, was observed for each peptide. Amino acid sequence-determining fragment ion were produced by CID and collected by MS/MS for the three linear peptides, and also for the two disulfide-bond-containing peptides in their unreduced and dithiothreitol (DTT)-reduced forms. The detection level for the [M + H]+ ion of DTLET was ca. 3 pmol; and the stabilities of the CTAP and ICI analogs in plasma were studied.     

Resource utilization in traumatic brain injury: the role of magnetic resonance imaging

A number of potential matrix candidates were investigated with regard to the importance of the pH in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) analysis of non-covalently bound protein complexes. The matrices examined were 2,5-dihydroxybenzoic acid (DHB), 4-hydroxy-alpha-cyanocinnamic acid (HCCA), 2-aminonicotinic acid (ANA), 4-nitroaniline (NA), 2-amino-4-methyl-5-nitropyridine (AMNP) and 3-hydroxypicolinic acid (HPA). In solution these matrix compounds permitted the preparation of MALDI samples at pH in the range 2-7. Among the matrices tested, complex formation, by specific non-covalent interactions, could only be observed when HPA (pH 3.8) was used as the matrix for the MALDI analysis. Under these conditions, specific non-covalent complex formation of recombinant streptavidin and glutathione-S-transferases were observed but not for human hemoglobin. The MALDI spectra obtained with the neutral compounds ANA (pH 4.4), NA (pH 6.4) and AMNP (pH 7.1) as matrices contain only peaks of the subunit with no signal of the non-covalent bound complexes present. Considering the results obtained in this study with basic and acidified matrix materials, there does not appear to be a strong correlation between the pH of the matrix solution and the utility of a matrix for the analysis of non-covalently bound complexes.     

Structures of bacitracin A and isolated congeners: sequencing of cyclic peptides with blocked linear side chains by electrospray ionization mass spectrometry

The bacitracin antibiotic complex consists principally of bacitracin A, a peptide antibiotic containing seven amino acid residues in a ring and five amino acid residues in a blocked side chain, together with a mixture of minor components presumably related but of unknown structures. A preparative high-performance liquid chromatographic method was developed for isolating the minor components A2, B1 and B2 which were then characterized by amino acid analysis, exact mass fast atom bombardment (FAB) mass spectrometry, FAB tandem mass spectrometry (MS/MS) and electrospray ionization (ESI) mass spectrometry. For bacitracins A (MW 1421), A2 (MW 1421), B1a (MW 1407), B1b (MW 1407), B2 (MW 1407) and F (MW 1419), the side chain sequences were determined by ESI MS/MS and ESI nozzle-skimmer collision-induced dissociation (CID) mass spectrometry and the ring sequences elucidated by ESI nozzle-skimmer CID MS/MS. Relative to bacitracin A, bacitracin A2a has the modified isoleucine residue at position 1 replaced by a modified allo-isoleucine residue, bacitracin B1a has the isoleucine residue at position 8 replaced by a valine residue, bacitracin B1b has the isoleucine residue at position 5 replaced by a valine residue and bacitracin B2 has the modified isoleucine residue at position 1 replaced by a modified valine residue. FAB tandem mass spectra were shown to be consistent with the above structural assignments for the isolated bacitracin components. Structures were also proposed for the trace bacitracin components C1 (MW 1393) and D1 (MW 1379) using ESI MS/MS data obtained from the analysis of the bacitracin complex without isolation.     

Physiological role of human placental growth hormone

Described herein are the fragmentation pathways of kanamycin A and its 6'-N- and 1-N-acyl derivatives, as well as the determination of their positional isomers by FABMS and ESIMS in combination with tandem mass spectrometry. The presence or absence of key ions and the difference in abundance of common ions are correlated with the position of the substitution.     

Rapid identification of comigrating gel-isolated proteins by ion trap-mass spectrometry

In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.     

Accurate mass determinations for the confirmation and identification of organic microcontaminants in surface water using on-line solid-phase extraction liquid chromatography electrospray orthogonal-acceleration time-of-flight mass spectrometry

The identification of polar microcontaminants in surface water is an important issue in environmental analysis. Liquid chromatography/mass spectrometry (LC/MS) is frequently applied for this purpose. However, even in combination with tandem mass spectrometry (MS/MS), unambiguous identification of the compounds detected is often difficult. The potential of an alternative strategy, based on the ability of an orthogonal-acceleration time-of-flight mass spectrometer to routinely perform accurate mass determination at 10 ppm in on-line LC/MS, is explored. On-line solid-phase extraction LC electrospray orthogonal-acceleration time-of-flight mass spectrometry is shown to enable the determination of pesticides from various compound classes in surface water in the concentration range of 0.1 to 10 micrograms/L. In addition, the ability to discriminate and unambiguously identify pesticides in mixtures of isobaric and/or isomeric compounds is investigated.     

Determination of hepatotoxic pyrrolizidine alkaloids by on-line high performance liquid chromatography mass spectrometry with an electrospray interface

The present study describes the determination of two different types of hepatotoxic pyrrolizidine alkaloids (PAs) and also distinguishing the hepatotoxic PAs from non-toxic ones by both in-source collision-induced dissociation high performance liquid chromatography mass spectrometry (CID-HPLC/MS) and HPLC/MS/MS (CID in the collision cell), using electrospray ionization. The mass spectra provided molecular ions and characteristic fragment ions, which could be used readily for a rapid identification of different types of PAs. Applications of both in-source CID-HPLC/MS and HPLC/MS/MS analytical methods were successful for the determination of PAs in blood samples obtained from rats dosed with PAs and in the PA-containing plant. The results demonstrated that the developed HPLC/MS methods with two different CID techniques provided a very simple and rapid analysis for an unequivocal diagnosis of PA poisoning and for definitive identification of PAs in plants or herbal medicines.     

Crystal structure of Boc-LAla-deltaPhe-deltaPhe-deltaPhe-deltaPhe-NHMe: a left-handed helical peptide

We describe the application of immunoaffinity extraction and mass spectrometry to the analysis of Ty1 Gag protein in lysates of Saccharomyces cerevisiae. A magnetic bead-conjugated monoclonal antibody was used to achieve selective extraction, the specificity of which was established by matrix-assisted laser desorption/ionization mass spectrometric (MS) analysis of an extract of the lysate of cells overexpressing the Ty1 Gag protein. MS analysis of similar extracts of lysates following tryptic hydrolysis confirmed selective extraction of the epitope-containing peptide fragment. Sufficient sensitivity was achieved to allow the application of this approach to the analysis of lysates of wild-type cells. Furthermore, the sequence of the epitope-containing peptide was confirmed by electrospray-tandem MS. To our knowledge, this constitutes the first report of the application of immunoaffinity extraction and tandem MS analysis to the characterization of an antigen recovered from a complex cellular system.     

An Intercomparison Study of Analytical Methods for the Determination of Magnesium in Low Alloy Steel

In an intercomparison study three low alloy steel materials were analyzed on their content of the trace element Mg, and five different analytical techniques were used, namely spark‐OES, inductively coupled plasma optical emission spectrometry (ICP‐OES), inductively coupled plasma time of flight mass spectrometry (ICP‐TOFMS), inductively coupled plasma quadrupole mass spectrometry (ICP‐QMS), and glow discharge mass spectrometry (GD‐MS). Solid steel discs were used for analysis with spark‐OES and GD‐MS. For the analyses with ICP‐OES, ICP‐TOFMS, and ICP‐QMS steel chips were wet‐digested in aqua regia, and the wet‐digestion was performed either in polypropylene tubes placed in a heating block or in Teflon pressure vessels using a microwave assisted system. The Mg concentrations obtained for the three steel materials were: 2.0, 2.8, and 10.3 µg g^?1, respectively, and the spread in results was acceptable, giving RSD values in the range of 20–30%.     

CD45RC isoforms define two types of CD4 memory T cells, one of which depends on persisting antigen

A method to directly identify proteins contained in mixtures by microcolumn reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) is studied. In this method, the mixture of proteins is digested with a proteolytic enzyme to produce a large collection of peptides. The complex peptide mixture is then separated on-line with a tandem mass spectrometer, acquiring large numbers of tandem mass spectra. The tandem mass spectra are then used to search a protein database to identify the proteins present. Results from standard protein mixtures show that proteins present in simple mixtures can be readily identified with a 30-fold difference in molar quantity, that the identifications are reproducible, and that proteins within the mixture can be identified at low femtomole levels. Based on these studies, methodology has been developed for direct LC/MS/MS analysis of proteins enriched by immunoaffinity precipitation, specific interaction with a protein-protein fusion product, and specific interaction with a macromolecular complex. The approach described in this article provides a rapid method for the direct identification of proteins in mixtures.     

Total structures of colistin minor components

Structural characterization of the colistin (CL) components were carried out using Frit-fast atom bombardment liquid chromatography/mass spectrometry (Frit-FAB LC/MS), tandem mass spectrometry (MS/MS) and the amino acid analysis proposed by MARFEY, and the total structures of 4 minor components including the absolute configuration of the constituent amino acids were proposed. The structures of the minor components were the same as those of the main component colistin A or B except that L-leucine is replaced by L-valine or L-isoleucine.     

Activities of the enzymes participating in purine and free-radical metabolism in cancerous human colorectal tissues

Liquid chromatography-pneumatically assisted electrospray mass spectrometry with negative ionization has been used for the determination of acidic herbicides in ground water. Eighteen pesticides or pesticide degradation products belonging to several different groups of acidic herbicides (phenoxy acids, sulfonylureas, phenols, etc.) were covered in the study. Optimization of electrospray inlet conditions is described as well as results from investigations of the linearity of the detector response. Conditions for tandem mass spectrometry (MS-MS) detection of characteristic daughter ions formed by collision-induced dissociation (CID) of the parent ion are described and a comparison of obtainable instrument detection limits by single MS and MS-MS was made. Detection limits using MS in the selected ion monitoring (SIM) mode were generally in the order of 1 microgram/l or below, whereas detection limits were three-four times higher using MS-MS detection. A principle of analysis is proposed based on single quadrupole MS as a method for quantitative determination followed by verification of positive findings by CID MS-MS. Application of the method for detecting acidic herbicides residues in a "real-world" ground water sample is demonstrated.     

Mass spectrometric analysis of tetracycline antibiotics in foods

We will review recent developments in mass spectrometric analysis of tetracycline antibiotics (TCs) in foods. The mass spectrometric techniques discussed are as follows: the collision-activated decomposition mass-analysed ion kinetic energy spectrometry (CAD MIKES), thin-layer chromatography (TLC)- fast atom bombardment (FAB) mass spectrometry (MS), particle beam (PB) liquid chromatography (LC)-MS, LC-fit FAB MS, thermospray (TSP) LC-MS, atmospheric chemical ionization (APCI) LC-MS and tandem electrospray (ESI) LC-MS. Their advantages and limitations are described in the confirmation of TCs in foods: CAD MIKES can confirm TCs with high sensitivity; however, its practical application is questionable because of uncommon instrumentation. TSP has a problem in reproducibility of the mass spectrum. Although TLC-FAB-MS can be applied to any kind of samples, it cannot be used for the quantitative analysis. LC-frit FAB-MS is a useful technique for the confirmation of TCs in honey, but it cannot be applied to animal tissues because of a lack of sensitivity. PB negative chemical ionization, APCI, and ESI-MS-MS can reliably confirm TCs in foods with good reproducibility.     

Investigation of the covalent modification of the catalytic triad of human cytomegalovirus protease by pseudo-reversible beta-lactam inhibitors and a peptide chloromethylketone

An investigation into the interaction between human cytomegalovirus (HCMV) protease and several beta-lactams, with characterization of the resulting acylenzymes using mass spectrometry, is reported. The time dependence of the inhibitors is highlighted by making comparisons of values obtained for inhibition and acylation. Analysis of inactivated HCMV protease revealed a beta-lactam: protease stoichiometry of 1. Subsequent enzymatic digestion with trypsin, peptide mapping using liquid chromatography coupled with electrospray ionization mass spectrometry and sequencing by nanoelectrospray tandem mass spectrometry (NanoES-MS/MS) allowed the identification of the site of covalent modification and confirmed Ser 132 as the active site hydroxyl nucleophile. Further, treatment of the protease with a peptide chloromethylketone and sequence analysis using NanoES-MS/MS of the alkylated enzyme confirmed His 63 as the active site imidazole nucleophile.     

Determination of four anabolic steroid metabolites by gas chromatography/mass spectrometry with negative ion chemical ionization and tandem mass spectrometry

A gas chromatography/mass spectrometry method is described which uses negative ion chemical ionization and tandem mass spectrometry for the determination of anabolic steroid metabolites. Four anabolic steroid metabolites to be derivatized by Pentafluoropropionic anhydride (PFPA) were determined using gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization (NCI) and NCI/MS/MS. The repeatability and reproducibility of this procedure were in the range of 5.3-9.7% and 6.1-10.2%, respectively. This method of derivatization with PFPA for NCI and NCI/MS/MS was useful to determine four metabolites of nandrolone, dromostanolone, methenolone and boldenone. The derivatized metabolites of boldenone could be detected to 2 ppb and the other three steroids could be detected to 25 ppb in urine at a signal-to-noise ratio of S/N = 3.     

An automated liquid chromatography/mass spectrometry system coupled on-line with microdialysis for the in vivo analysis of contrast agents

An automated high-performance liquid chromatography (HPLC)/mass spectrometry instrument coupled on-line with a microdialysis probe is described for the analysis of contrast agents in biological fluids. This system is used for pharmacokinetic and structure elucidation studies of contrast agents used in X-ray and magnetic resonance imaging investigations. Additionally an LC/MS system for the direct analysis of urine, that obviates the need for conventional sample clean-up, is described.