Microbore Liquid Chromatography Research Articles

Electrospray ionization developed by Lidija Gall's group

This historical review is dedicated to Prof. Lidija Gall. She was awarded the Thomson medal (2022) for her contribution to the development of electrospray ionization mass spectrometry in the early 1980s in Leningrad, Soviet Union. We describe the rationale and provide historical references that highlight her way to the discovery of a method of solvated ion extraction at atmospheric pressure (SIEAP), known by the Russian abbreviation ЭРИАД (ERIAD), which is the direct analogue of electrospray ionization developed in the West. Even at the early prototype stage, ERIAD was able to determine the intact masses of a broad scope of polar biomolecules including peptides, small proteins, nucleotides, carbohydrates, and prostanglandins. The ERIAD source was directly coupled to microbore liquid chromatography and was applied to analyze protein digests, to characterize the substrate specificity of proteolytic enzymes, to mapping of mutation sites in physiologically important proteins and also to increase the confidence in protein sequence determinations. In inorganic chemistry, ERIAD was successfully applied to analyze the solutions of rare-earth metals and metal complexes. Altogether, a set of experimental studies and a broad scope of applications contributed to better understanding of the ionization mechanism and the methods needed to acquire and interpret ERIAD spectra. The method was first implemented on a magnetic sector mass spectrometer and then was adopted to a variety of mass analyzers and mobility separators in collaboration with Russian mass spectrometry groups.

Highly stable peptide adducts from hard keratins as biomarkers to verify local sulfur mustard exposure of hair by high-resolution mass spectrometry

In the recent past, the blister agent sulfur mustard (SM) deployed by the terroristic group Islamic State has caused a huge number of civilian and military casualties in armed conflicts in the Middle East. The vaporized or aerolized agent might be inhaled and have direct contact to skin and hair. Reaction products of SM with plasma proteins (adducts) represent well-established systemic targets for the bioanalytical verification of exposure. The SM-derived hydroxyethylthioethyl (HETE)-moiety is attached to nucleophilic amino acid side chains and allows unambiguous adduct detection. For shipping of common blood and plasma samples, extensive packaging rules are to be followed as these matrices are considered as potentially infectious material. In contrast, hair is considered as non-infectious thus making its handling and transportation much less complicated. Therefore, we addressed this matrix to develop a procedure for bioanalytical verification. Following optimized lysis of SM-treated human scalp hair and pepsin-catalyzed proteolysis of adducts of keratin type I and II, microbore liquid chromatography–electrospray ionization high-resolution tandem-mass spectrometry (µLC–ESI MS/HR MS) was used to detect three alkylated keratin-derived biomarker peptides: AE(-HETE)IRSDL, FKTIE(-HETE)EL, and LE(-HETE)TKLQF simultaneously. All bear the HETE-moiety bound to a glutamic acid residue. Protein adducts were stable for at least 14 weeks at ambient temperature and contact to air, and were not affected by washing the hair with shampoo. The biomarker peptides were also obtained from beard, armpit, abdominal, and pubic hair. This is the first report introducing stable local peptide adduct biomarkers from hair, that is easily accessible by a non-invasive sampling process.

The influence of injection volume on efficiency of microbore liquid chromatography columns for gradient and isocratic elution

The injection volume and the associated column volume overload is one of the most common issues in miniaturized chromatography. The injection volume should not exceed 10% of the effective column volume. A further reduction of the injection volume leads to an increase in chromatographic efficiency. However, the signal intensity must be above a certain threshold to generate a chromatographic peak that can be detected. Therefore, the injection volume has to be optimized to reach the ideal balance between chromatographic efficiency and sensitivity. This study examined the general influence of the injection volume for both isocratic and gradient elution, depending on the retention factor and peak standard deviation. For this purpose, substances of different polarity were selected to represent a broad elution spectrum. Besides the model analyte naphthalene, these were mainly pharmaceuticals. For all measurements a microbore column with an ID of 300 µm and packed with 1.9 μm fully porous particles was used. For isocratic elution, the injection volume was varied between 4 and 16% of the effective column volume. The retention factors were adjusted between 2 and 10. For gradient elution, the injection volume was varied between 4 and 160% of the effective column volume. The observed effects were further investigated using the gradient kinetic plot theory. In isocratic elution, a loss in plate height up to 50% was observed for components that elute near the void time. A significant reduction of the chromatographic efficiency was noticed up to a retention factor of 4. In gradient elution, a reduction in peak capacity could only be observed if the injection volume exceeded 40% of the effective column volume. For some substances, only a slight loss in peak capacity was noticed even with a volume overload of 160%.

Determination of unbound hesperetin in rat blood and brain by microdialysis coupled to microbore liquid chromatography

A rapid and sensitive microbore high-performance liquid chromatography (HPLC) coupled to microdialysis technique for simultaneous determination of unbound hesperetin in rat blood and brain has been developed. The HPLC assay was carried out using a microbore reversed-phase C-18 column (100 × 1 mm I.D., 5 μm particle size). The mobile phase used was acetonitrile-methanol-20 mM monosodium phosphate (pH 5.3) (40:10:50, v/v/v) for hesperetin at a flow-rate of 0.05 mL/min. The assay was performed by monitoring the wavelength of maximum UV absorbance at 288 nm. Microdialysis probes were inserted into the jugular vein/right atrium and brain striatum of male Sprague-Dawley rats. Hesperetin (50 mg/kg, i.v., n=6) was administered via the femoral vein, and microdialysates were collected from both sites and assayed by the validated microbore scale HPLC method. Pharmacokinetic parameters were calculated from the corrected data for dialysate concentrations of hesperetin versus time.

S – Cell proliferation and carcinogenesis: bad luck and the environment

Exposure to toxic organophosphorus pesticides (OPP) represents a serious problem in the public healthcare sector and might be forced in terroristic attacks. Therefore, reliable verification procedures for OPP-intoxications are required for forensic, toxicological and clinical reasons. We developed and optimized a toolbox of methods to detect adducts of human serum albumin (HSA) with OPP considered as long-term biomarkers. Human serum was incubated with diethyl-oxono and diethyl-thiono pesticides for adduct formation used as reference. Afterwards serum was subjected to proteolysis using three proteases separately thus yielding phosphorylated tyrosine residues (Y*) detected as single amino acid (pronase), as hexadecapeptide LVRY*411TKKVPQVSTPTL (pepsin) and as the tripeptide Y*411TK (trypsin), respectively. Adducts were analyzed via microbore liquid chromatography coupled to electrospray ionization (μLC-ESI) and tandem-high-resolution mass spectrometry (MS/HR MS).Using paraoxon-ethyl as model OPP for adduct formation, methods were optimized with respect to MS/HR MS-parameters, protease concentrations and incubation time for proteolysis. HSA-adducts were found to be stable in serum in vitro at +37 °C and −30 °C for at least 27 days and resulting biomarkers were stable in the autosampler at 15 °C for at least 24 h. Limits of identification of adducts varied between 0.25 μM and 4.0 μM with respect to the corresponding pesticide concentrations in serum. Applicability of the methods was proven by successful detection of the adducts in samples of OPP-poisoned patients thus demonstrating the methods as a reliable toolbox for forensic and toxicological analysis.

A toolbox for microbore liquid chromatography tandem-high-resolution mass spectrometry analysis of albumin-adducts as novel biomarkers of organophosphorus pesticide poisoning

Exposure to toxic organophosphorus pesticides (OPP) represents a serious problem in the public healthcare sector and might be forced in terroristic attacks. Therefore, reliable verification procedures for OPP-intoxications are required for forensic, toxicological and clinical reasons. We developed and optimized a toolbox of methods to detect adducts of human serum albumin (HSA) with OPP considered as long-term biomarkers. Human serum was incubated with diethyl-oxono and diethyl-thiono pesticides for adduct formation used as reference. Afterwards serum was subjected to proteolysis using three proteases separately thus yielding phosphorylated tyrosine residues (Y*) detected as single amino acid (pronase), as hexadecapeptide LVRY*411TKKVPQVSTPTL (pepsin) and as the tripeptide Y*411TK (trypsin), respectively. Adducts were analyzed via microbore liquid chromatography coupled to electrospray ionization (μLC-ESI) and tandem-high-resolution mass spectrometry (MS/HR MS).Using paraoxon-ethyl as model OPP for adduct formation, methods were optimized with respect to MS/HR MS-parameters, protease concentrations and incubation time for proteolysis. HSA-adducts were found to be stable in serum in vitro at +37 °C and −30 °C for at least 27 days and resulting biomarkers were stable in the autosampler at 15 °C for at least 24 h. Limits of identification of adducts varied between 0.25 μM and 4.0 μM with respect to the corresponding pesticide concentrations in serum. Applicability of the methods was proven by successful detection of the adducts in samples of OPP-poisoned patients thus demonstrating the methods as a reliable toolbox for forensic and toxicological analysis.

Characterization of peak capacity of microbore liquid chromatography columns using gradient kinetic plots

The performance of micro-liquid chromatography columns with an inner diameter of 0.3mm was investigated on a dedicated micro-LC system for gradient elution. Core-shell as well as fully porous particle packed columns were compared on the basis of peak capacity and gradient kinetic plot limits. The results for peak capacity showed the superior performance of columns packed with sub-2μm fully porous particles compared to 3.0μm fully porous and 2.7μm core-shell particles within a range of different gradient time to column void time ratios. For ultra-fast chromatography a maximum peak capacity of 16 can be obtained using a 30s gradient for the sub-2μm fully porous particle packed column. A maximum peak capacity of 121 can be achieved using a 5min gradient. In addition, the influence of an alternative detector cell on the basis of optical waveguide technology and contributing less to system variance was investigated showing an increased peak capacity for all applied gradient time/column void time ratios. Finally, the influence of pressure was evaluated indicating increased peak capacity for maximum performance whereas a limited benefit for ultra-fast chromatography with gradient times below 30s was observed.

Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis.

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300μm has been investigated using a dedicated micro-liquid chromatography system. Fully porous, core-shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub-2μm fully porous as well as the 2.7μm core-shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56-2.69) before correction for extra-column contribution compared to normal-bore columns. Moreover, the influence of extra-column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub-2μm fully porous particle packed column for ultra-fast liquid chromatography.

EGb761 Co-administrated with FK506 Offer no More Neuroprotective Efficacy than EGb761 Alone during Focal Cerebral Ischemia and Reperfusion Monitored by Microdialysis Coupled with Microbore Liquid Chromatography

Accumulated dopamine (DA) is believed to be detrimental to the brain. EGb761 and FK506 have been reported to possess neuroprotective efficacy. This study attempted to investigate the metabolic pathway of dopamine and to realize whether the synergistic effect would be achieved when EGb761 co-administrated with FK506. Dynamic changes of dopamine and its metabolites including 3, 4-dihydroxyphenylacetate (DOPAC), homovanillic acid (HVA) and 3-methoxytyramine (3-MT) in the striatum were measured. Histological technique of 2, 3, 5-triphenyltetrazolium chloride (TTC) staining was used for assessing the infarction volume. Data indicated that an approximately 80, 90, and 180 fold increases of the dopamine level was seen in the group of EGb761, control, and combination (EGb761 plus FK506) during ischemia. Interestingly, no significant changes in the level of DOPAC, HVA, and 3MT were observed among these groups. Histological evidence showed that the mean total infarction size was significantly elevated in the combination group when compared with the control group (P<0.05). Importantly, compared with the control group, the infarction volume at the slices of 4mm on the striatum was significantly higher in the combination group (P<0.05). Accordingly, it seems possible that cerebral ischemia may lead to more accumulated dopamine; meanwhile, most of the accumulated dopamine may be rapidly catabolited through a less energy consumed route and eventually to form more alternative metabolites other than DOPAC, 3-MT and HVA. In addition, EGb761 co-administrated with FK506 offers no more neuroprotective efficacy than EGb761used alone during cerebral ischemia.

Microbore liquid chromatography ultraviolet detection for quantification of total peptide amount and its application for assessing sample quality in shotgun proteome analysis of hundreds of cells

Mass spectrometric profiling of the proteome of a small number of cells requires not only a sensitive instrument for protein/peptide separation and detection, but also a robust sample preparation protocol to process a very small amount of proteins (<1μg) present in few cells. We have developed and evaluated the performance of a microbore liquid chromatography (LC) UV detection system for quantifying the total amount of peptides in a shotgun proteome analysis workflow that is tailored for the analysis of hundreds of cancer cells. Upon the sample injection into a 1-mm-diameter reversed phase column, a step-gradient was used to first remove salts and other impurities and then elute the peptides quickly without much separation. The UV absorbance of eluted peptides at 214nm was used for peptide quantification with the aid of a calibration curve of a tryptic digest of a mixture of four standard proteins. Two linear calibration regions could be obtained in the peptide amount ranging from 0.03μg to 0.3μg and from 0.6μg to 5μg. The limit of quantification (LOQ) was determined to be 30ng (or 39ng in the linear calibration range). However, the presence of background proteins, mainly keratins, introduced during the sample preparation process was found to be the limiting factor in quantifying a lower amount of peptides from a cell lysate digest. With background absorbance from the digest of contaminant proteins in a solution, the LOQ was found to be 200ng. This nondestructive microbore LC-UV method should be useful in assessing sample quality during the development and applications of an efficient sample preparation method for proteome analysis of a small number of cells. As an example, this method was used for quantifying the peptides generated from breast cancer MCF-7 cell extracts with a limited number of cells: 250, 500 and 1000 cells. Using capillary LC quadrupole time-of-flight mass spectrometry, 81–126, 122–154 and 256–282 proteins could be identified from 250, 500, and 1000 cells, respectively, in duplicate experiments. This method was also applied for the analysis of biological triplicate samples of MCF-7 cells. The average numbers of peptides and proteins detected from the experimental triplicate analyses of biological triplicate samples were 400±71 (9 datasets) and 124±14, respectively, from 250 cells, and 531±44 and 162±16, respectively, from 500 cells.

Quantitative analysis of PD 0332991 in mouse plasma using automated micro-sample processing and microbore liquid chromatography coupled with tandem mass spectrometry

In the oncology therapeutic area, the mouse is the primary animal model used for efficacy studies. Often with mouse pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) studies, less than 20 μL of total plasma sample volume is available for bioanalysis due to the small size of the animal and the need to split samples for other measurements such as biomarker analyses. The need to conduct automated “small volume” sample processing for quantitative bioanalysis has therefore increased. An automated fit for purpose protein precipitation (PPT) method using a Hamilton MicroLab Star (Reno, NV, USA) to support mouse PK and PK/PD studies for an oncology drug candidate PD 0332991, (a specific inhibitor of cyclin-dependent kinase 4 (CDK-4) currently in development) for processing “small volumes” was developed. The automated PPT method was achieved by extracting and processing 10 μL out of a minimum sample volume of 15 μL plasma utilizing the Hamilton MicroLab Star. A 96-conical shallow well plate by Agilent Technologies, Inc (Wilmington, DE, USA) was the labware of choice used in the automated Hamilton “small volume” method platform. Analyses of a 10 μL plasma aliquot from 15 μL of plasma study samples were conducted by both automated and manual PPT method. All plasma samples were quantitated using a Sciex API 4000 triple quadrupole mass spectrometer coupled with an Eksigent Express HT Ultra HPLC system. The chromatography was achieved using an Agilent microbore C 18 Extend, 1.0 × 50 mm, 3.5 μm column at a flow rate of 0.150 mL/min with a total run time of 1.8 min. Accuracy and precision of standard and QC concentration levels were within 90–107% and <14%, respectively. Calibration curves were linear over the dynamic range of 1.0–1000 ng/mL. PK studies for PD 0332991 were conducted in female C3H mice following intravenous administration at 1 mg/kg and oral administration at 2 mg/kg. PK values such as area under curve (AUC), volume of distribution (Vd), clearance (Cl), half life ( T 1/2) and bioavailability ( F%) demonstrated less than 11% difference between the automated Hamilton and manual PPT methods. The results demonstrate that the automated Hamilton PPT method can accurately and precisely aliquot 10 μL of plasma from 15 μL or larger volume plasma samples. The fit for purpose Hamilton PPT method is suitable for routine analyses of plasma samples from micro-sampling PK and PK/PD samples to support discovery studies.

Microbore liquid chromatography with UV detection to study the in vivo passage of compound 21, a non-peptidergic AT 2 receptor agonist, to the striatum in rats

A microbore liquid chromatography method coupled to UV detection was developed and validated in order to monitor the passage of compound 21 (C 21), a non-peptide angiotensin II type 2 receptor agonist, to the striatum of rats. For this purpose, sampling from the striatum was performed using the in vivo microdialysis technique. Separations were performed on a C 18 microbore (1 mm i.d.) column using gradient elution. The retention time for C 21 was found to be 6.3 min. The calibration curve was linear between 10 and 200 ng/ml with a correlation coefficient ≥0.999. The limit of detection and the limit of quantification were 3 and 10 ng/ml respectively. The intra-day and the inter-day precision (RSD%) ranged between 0.5 and 4.6% with an average recovery of 101.5 ± 10.0% (mean ± SD, n = 15). In vivo experiments were performed on rats to measure the concentration of C 21 in striatal dialysates after intraperitoneal (10 or 50 mg/kg) or intravenous injection (10 mg/kg or 20 mg/kg) of C 21 and suggest minimal passage of the compound to the striatum.

Investigating the possible use of a tetra (hydroxyphenyl) porphyrin as a fluorescence probe for the supramolecular detection of phospholipids

The present work explores the interest of using the 5,10,15,20-tetrakis(3-hydroxyphenyl)-21H,23H-porphine ( m-THPP), a commercially available porphyrin, as a polarity probe for the detection of phospholipids. Feasibility of the process was first tested by direct fluorimetry and it was established that emission enhancement in the presence of lipids was maximum for a methanol content of 15% (v/v), a neutral pH (6–8) and a probe concentration of 2×10 −7 M in the working solution. Detection with m-THPP in these conditions presents higher response factor but shorter linear range than diphenylhexatriene (DPH), the reference dye for the supramolecular detection of lipids. Insights on the mechanism of m-THPP–lipid association demonstrated that, at high lipid concentrations, porphyrin is associated to lipid assemblies in monomer form and located in the interior of bilayer; in low lipid content, porphyrin aggregates coexist with the monomer. Finally it was assessed that the method can be successfully coupled with micro-bore liquid chromatography.

Versatile Capillary Column Temperature Control Using a Thermoelectric Array Based Platform

A new direct contact platform for capillary column precise temperature control based upon the use of individually controlled sequentially aligned Peltier thermoelectric units is presented. The platform provides rapid temperature control for capillary and microbore liquid chromatography columns and allows simultaneous temporal and spatial temperature programming. The operating temperature range of the platform was between 15 and 200 °C for each of 10 aligned Peltier units, with a ramp rate of approximately 400 °C/min. The system was evaluated for a number of nonstandard capillary based applications, such as the direct application of temperature gradients with both linear and nonlinear profiles, including both static column temperature gradients and temporal temperature gradients, and the formation of in-capillary monolithic stationary phases with gradient polymerization through precise temperature control.

Identification of very-long-chain polyunsaturated fatty acids from Amphidinium carterae by atmospheric pressure chemical ionization liquid chromatography–mass spectroscopy

A method is described for the enrichment of very-long-chain polyunsaturated fatty acids (VLCPUFAs) from total fatty acids of Amphidinium carterae and their identification as picolinyl esters by means of microbore liquid chromatography–mass spectrometry with atmospheric pressure chemical ionization (LC–MS/APCI). The combination of argentation TLC and LC–MS/APCI was used to identify unusual VLCPUFAs up to hexatriacontaoctaenoic acid. Two acids, 36:7n-6 and 36:8n-3, were also synthesized to unambiguously confirm their structure. The possibilities of VLCPUFAs biosynthesis are proposed.

Insulins in equine urine: qualitative analysis by immunoaffinity purification and liquid chromatography/tandem mass spectrometry for doping control purposes in horse‐racing

Insulin is a peptide hormone consisting of two peptide chains (A- and B-chain) that are cross-linked by two disulfide bonds. To obtain improved pharmacokinetic onset of action profiles of insulin treatment in diabetic patients, recombinant long-, intermediate-, and rapid-acting insulin analogs are produced, in which the C-terminal end of the B-chain plays an especially important role.A review of the veterinary literature reveals the low prevalence of equine type I diabetes mellitus, which indicates that the therapeutic use of insulin in racing horses is unlikely. Although there is no unequivocal evidence of an overall performance-enhancing effect of insulin, in human sports the misuse of insulin preparations is reported among elite athletes. The desired effects of insulin include the increase of muscular glycogen prior to sports event or during the recovery phase, in addition to a chalonic action, which increases the muscle size by inhibiting protein breakdown. In the present study urinary insulin was detected in equine samples and differences between equine insulin, human insulin, as well as rapidly acting recombinant insulin variants were examined. The method was based on sample purification by solid-phase extraction (SPE) and immunoaffinity chromatography (IAC), and subsequent analysis by microbore liquid chromatography (LC) and tandem mass spectrometry (MS/MS) using top-down sequencing for the determination of various insulins. Product ion scan experiments of intact proteins and B-chains enabled the differentiation between endogenously produced equine insulin, its DesB30 metabolite, human insulin and recombinant insulin analogs, and the assay allowed the assignment of individual product ions, especially those originating from modified C-termini of B-chains.

Development of novel guanidino‐labelling derivatisation (GLaD) reagents for liquid chromatography/matrix‐assisted laser desorption/ionisation analysis

A new generation of guanidino-labelling (GLaD) reagents were developed for quantitative proteomics using offline microcapillary liquid chromatography (LC) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). In order to reduce the unwanted overlapping between the isotopic envelopes of the two differentially labelled peptide ions, a novel synthetic route was described for production of both (13)C- and (15)N-containing isotopomers of N,O-dimethylisourea. The use of these types of isotopes has no deleterious effect on the retention times of both differentially labelled peptides during offline microbore reversed-phase LC. In addition, the possibility to incorporate a mass difference of 4 Da can be exploited during post-source decay analysis to generate product ion spectra in which fragment ions containing the modifications appear as doublets in the corresponding product ion spectra, thus facilitating identification of the C-terminal fragment ions.

A study of reproducibility of guanidination–dimethylation labeling and liquid chromatography matrix-assisted laser desorption ionization mass spectrometry for relative proteome quantification

The combination of dimethylation after gu anidination (2MEGA) isotope labeling with microbore liquid chromatography (LC)-matrix-assisted laser desorption ionization (MALDI) MS and MS/MS [C. Ji, N. Guo, L. Li, J. Proteome Res. 4 (2005) 2099] has been reported as a promising strategy for abundance ratio-dependent quantitative proteome analysis. A critical step in using this integrated strategy is to set up the abundance ratio threshold of peptide pairs, above which the peptide pairs are used for quantifying and identifying the protein that is considered to be differentially expressed between two different samples. The threshold is determined by technical variation (i.e., the overall abundance ratio variation caused by the experimental process including sample workup, MS analysis and data processing) as well as biological variation (i.e., the abundance ratio variation caused by the biological process including cell growth), which can be defined and assessed by a coefficient of variation (CV). We have designed experiments and measured three different levels of variations, starting with the same membrane protein preparation, the same batch of cells and three batches of cells from the same cell line grown under the same conditions, respectively. It is shown that technical variation from the experimental processes involved in 2MEGA labeling LC-MALDI MS has a CV of <15%. In addition, the measured biological variation from cell growth was much smaller than the measured technical variation. From the studies of the occurrence rate of outliers in the distribution of the abundance ratio data within a comparative dataset of peptide pairs, it is concluded that, to compare the proteome changes between two sets of cultured cells without the use of replicate experiments, a relative abundance ratio of greater than 2 X or less than 0.5 X ( X is the average abundance ratio of the dataset) on peptide pairs can be used as a stringent threshold to quantify and identify differentially expressed proteins with high confidence.

Comprehensive analysis of glycated human serum albumin tryptic peptides by off‐line liquid chromatography followed by MALDI analysis on a time‐of‐flight/curved field reflectron tandem mass spectrometer

Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end (AGE) product peptides, are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI based approaches the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatised peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Tandem mass spectrometric data suggest that the collision induced dissociation of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. The specific neutral losses derived from the activated glycated peptides can be used as signature for establishing the occurrence of glycation processes.

Identification of Microcystin Toxins from a Strain of Microcystis aeruginosa by Liquid Chromatography Introduction into a Hybrid Linear Ion Trap-Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

The cyclic heptapeptide microcystin toxins produced by a strain of Microcystis aeruginosa that has not been investigated previously were separated by liquid chromatography and identified by high-accuracy m/z measurements of their [M + H]+ ions and the fragment ions produced by collision-activated dissociation of the [M + H]+ ions. The cyanobacteria B2666 strain was cultured in a standard growth medium, and the toxins were released from the cells, extracted from the aqueous phase, and concentrated using standard procedures. The microcystins were separated by reversed-phase microbore liquid chromatography and introduced directly into a hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer with electrospray ionization. The known microcystins (MC) MC-LR, MC-LA, [MeSer7]MC-LR, MC-LL, MC-LF, and MC-L(Aba) were identified along with the two previously unreported structural variants [Asp3]MC-LA and [Asp3]MC-LL. In addition to the [M + H]+ ions, accurate m/z measurements were made of 12-18 product ions for each identified microcystin. The mean difference between measured and calculated exact m/z was less than 2 parts per million, which often allowed assignment of unique compositions to the observed ions. A mechanism is presented that accounts for an important collision-activated dissociation process that gives valuable sequence ions from microcystins that do not contain arginine. The analytical technique used in this work is capable of supporting fairly rapid and very reliable identifications of known microcystins when standards are not available and of most structural variants independent of additional information from other analytical techniques.