Molecular species of sphingomyelin: determination by high-performance liquid chromatography/mass spectrometry with electrospray and high-performance liquid chromatography/tandem mass spectrometry with atmospheric pressure chemical ionization

In a sphingomyelin-enriched sample of polar lipids from bovine milk, molecular species of intact sphingomyelin were separated by normal-phase high-performance liquid chromatography and detected by mass spectrometry (MS) for structural information. First, by using electrospray with positive ionization (ESI), protonated molecules ([M + H]+) were detected. Second, in atmospheric pressure chemical ionisation (APCI+), in-source fragmentation of sphingomyelin ions led to the formation of ceramide ions. With the ceramide ions as precursors, ions representative of both the long-chain base (LCB) parts and the fatty acid (FA) parts were detected in APCI-MS/MS via collision-induced decomposition (CID). Using this procedure, it was possible to determine the sphingomyelin molecular masses using ESI+ and then their respective LCB-FA combinations(s) using APCI+(-)MS/MS. At least 36 protonated molecules of intact sphingomyelin were detected in the bovine milk sample. The combinations found covered a range of molecular masses from 673 to 815 Da. The 12 most common protonated molecules (constituting approximately 90% of the total ion current in ESI) were composed of at least 25 different LCB-FA combinations. Saturated and unsaturated LCBs and FAs were detected in addition to hydroxy fatty acids. The most common LCBs were 16:1, 17:1, 18:1 and 19:1, whereas the most common FAs were 16:0, 22:0, 23:0 and 24:0. LCB-FA combinations of sphingomyelin from bovine brian, bovine erythrocytes and chicken egg yolk are also presented.     

The detection of the organic extractables in a biotech product by liquid chromatography on-line with electrospray mass spectrometry

We illustrated the ability of electrospray mass spectrometry (MS) to identify extractables from stoppers in biotech products. The advantages of using electrospray mass spectrometry (MS) as compared to the use of other traditional methods were also demonstrated, particularly, the capability of detecting the extractables from the mixtures of a peptide drug with its formulated excipients. MS alone could detect the antioxidant extractable, Butylated Hydroxytoluene (BHT), to the 0.5 ppm level. By using liquid chromatography (LC) on-line with MS, the detection of BHT improved at least to 0.1 ppm, depending on the LC column dimensions and injection volumes. Following aqueous buffer extraction, there were no detectable extractables nor chemical modifications of the peptide drug. When acetonitrile was used as the extraction solvent, we detected trace amounts of BHT (0.14 to 1.7 ppm) and a polymer with a molecular weight of 355 plus 44 mass unit extensions from the stopper. This unexpected polymer coeluted with BHT in LC could not be detected without on-line with MS. This incidence of coelution proved that the multidimensional approach, LC on-line with MS, is a powerful tool to characterize the extractables from the stopper and at the same time to assure the integrity of the drug substance.     

Specific detection and quantification of palmitoyl-stearoyl-phosphatidylserine in human blood using normal-phase liquid chromatography coupled with electrospray mass spectrometry

We have made an immunohistochemical study of the vomeronasal (VN) complex of 12-day-old rats to characterize the innervation of its blood vessels. The VN complex can be subdivided into rostral, middle and caudal segments, each one with a particular vascularization pattern. Several small vessels were associated with the rostral segment, whereas a large venous sinus ran along the middle and caudal segments. Immunostaining for alpha-smooth muscle actin demonstrated that the muscular sheath was asymmetric, with more cells layers in its lateral than in its medial walls. Nerves were demonstrated with antisera against protein gene product 9.5 (PGP), and against several molecules associated with specific classes of nerve fibers: the C-terminal peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (GAL), vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (NOS). The latter, was also studied with NADPH-diaphorase. Vascular associated fibers exhibited NOS-, CPON-, GAL-, CGRP-, SP- and VIP-immunoreactivity. Only the vessels of the rostral segment showed VIP-immunoreactive fibers. Each wall of the venous sinus exhibited different types of nerve fibers. CPON-, GAL-, CGRP- and SP-immunoreactive fibers concentrated in the medial wall, whereas NOS-immunoreactive ones concentrated in the lateral wall. This distribution of vascular fibers, plus the presence of sensory fibers exhibiting CGRP-, SP- and GAL-immunoreactivity within the pseudostratified epithelium of the VN tube, would be relevant to understand the operation of the pumping mechanism regulating influx and efflux from the VN tube.     

Rapid peptide mapping by reversed-phase liquid chromatography on nonporous silica with on-line electrospray time-of-flight mass spectrometry

Reversed-phase liquid chromatography (LC) using a nonporous silica support has been combined with electrospray (ES) time-of-flight (TOF) mass spectrometry (MS) for the fast separation and mass detection of peptides. Using this LC method, the resolution of a peptide mixture can be completed is less than 35 s. The resulting chromatographic peak widths are less than 1 s wide. Because of the unique nature of a TOF mass analyzer, complete mass spectra can be acquired at a rate which is sufficient to sample these narrow peaks. When compared with conventional LC, the same separation takes nearly 20 min to complete, and the signal-to-noise ratio observed in the total ion chromatogram is dramatically lower due to the influence of increased background noise in the mass spectra. The limit of detection for a low molecular weight peptide, Val-Pro-Leu, was found to be 6 pmol with the total ion chromatogram and 500 fmol with the reconstructed ion chromatogram. A peptide map of horse heart myoglobin, completed in 3.5 min, is shown as an example of the results which can be obtained from combining this fast LC method with fast ES/TOF/MS detection capability.     

Capillary liquid chromatography/electrospray mass spectrometry for the separation and detection of catechins in green tea and human plasma

The separation and detection of biologically active green tea catechins has been accomplished using capillary liquid chromatography/electrospray mass spectrometry (cLC/ESI-MS). Microscale determination (approximately 20 ng) of all six catechins in a green tea infusion, and the most extensively studied catechin, (-)epigallocatechin gallate (EGCG), in human plasma is demonstrated by cLC/ESI-MS with selected ion monitoring of protonated molecular ions. The overall quality of the analysis is shown to be dependent on the use of a capillary column with a deactivated, monomeric C18 stationary phase. The high chromatographic separation efficiency of this packed-capillary column, combined with the high sensitivity and selectivity afforded by the mass spectrometer as detector, provide a reliable approach to the analysis of picomolar quantities of these interesting compounds in complex matrices.     

Development of a nanoscale liquid chromatography/electrospray mass spectrometry methodology for the detection and identification of DNA adducts

In this work, the coupling of liquid nanochromatography to NanoFlow electrospray mass spectrometry was evaluated for the detection of DNA adducts. The NanoFlow ES LC/MS system was compared with the capillary and conventional ES LC/MS system by analyzing an in vitro reaction mixture resulting from the interaction of 2'-deoxyguanosine 5'-monophosphate with bisphenol A diglycidyl ether and by injecting 2'-deoxyadenosine. By using NanoFlow ES LC/MS, the mass sensitivity could be improved by a factor of 3300. Three different injection methods used in liquid nanochromatography, i.e., split, large-volume, and column-switching injections were compared in terms of sensitivity. Furthermore, NanoFlow ES LC/MS was used to detect 2'-deoxynucleotide adducts isolated from an in vitro mixture of calf thymus DNA and bisphenol A diglycidyl ether. Different 2'-deoxynucleotide adducts could be identified by monitoring typical product ions, diagnostic for the adducts.     

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.     

Analysis of Vero toxins 1 and 2 by high-performance liquid chromatography/electrospray ionization mass spectrometry

PURPOSE: To study the interaction between gabapentin (GBP) and high-protein meals, 12 patients with epilepsy were administered this drug both while in a fasting state and after a high-protein meal. METHODS: After having acquired their informed consent, the patients (suffering from partial complex seizures resistant to other anticonvulsants) were randomly assigned to 2 groups of 6 subjects. Each subject was treated in a fasting state with a single 400 (group A) or 800 (group B) mg GBP oral dose. After 24 h, the GBP dose regimen was repeated, but was given after a high-protein meal. Serum GBP concentrations were measured by LC-Mass at baseline and 0.5, 1, 2, 3, 5, 7, 9, 12, and 24 h. Saliva GBP concentrations were determined at baseline and 2, 4.8, and 12 h. GBP urinary excretion was determined at 0-4, 4-8, and 8-12 h intervals. The following kinetic parameters were calculated: area under the concentration time curve from zero time to 24 h after the dose, AUC 0-24 h; maximal serum concentration, Cmax; time to the maximal serum concentration, Tmax; absorption rate constant, ka; elimination rate constant, beta; elimination half-time, t1/2beta. Student's t test for paired data, with significance assigned at P < 0.05, was used. RESULTS: No statistically significant differences were seen in GBP serum or saliva concentrations or in its urinary excretion (both in A or B group) between fasting and after the high-protein meal. CONCLUSIONS: High-protein meals do not seem to interfere with oral disposition of GBP.     

Investigation of beta-amyloid peptide by on-line high-performance liquid chromatography coupled to electrospray ionization mass spectrometry

beta(1-39) amyloid peptide is one of the components of the cerebral amyloid deposits that are characteristic of Alzheimer's disease. Solid-phase synthesis of this peptide resulted in a fairly complex crude product containing both the target peptide and a number of side products. High-performance liquid chromatography coupled to electrospray ionization mass spectrometry allowed rapid and reliable identification of both the desired peptide and most of the side products which were found to have relative molecular masses above and below that of the target peptide.     

Sodium dodecyl sulphate removal from tryptic digest samples for on-line capillary liquid chromatography/electrospray mass spectrometry

An on-line microcolumn switching method was developed for the removal of sodium dodecyl sulphate (SDS) from tryptic digest samples. The system includes two micro-precolumns: a specific ionic detergent trapping column and a preconcentration column. Characterization of the proteinaceous samples, after isolation from the SDS, was performed by capillary liquid chromatography (LC) with UV absorption detection and electrospay mass spectrometry (ESI-MS). Loading and clean-up of the samples and regeneration of the detergent trapping column were performed at 50 microl min(-1), resulting in sample clean-up times of only 30 s. SDS-containing tryptic digested protein samples were directly applied to the micro-precolumns without any previous sample pretreatment. The developed microcolumn switching method permits the on-line analysis of small tryptic digest samples by capillary LC/ESI-MS in the presence of SDS. The method is completely automated and can be performed unattended. The maximum amount of SDS, in terms of loadability and breakthrough, were determined. Also studied were the selection of the loading and clean-up solvents and the recovery of the peptides. Chromatographic separations and mass spectral data confirmed the removal of SDS.     

Reverse-phase capillary high performance liquid chromatography/high performance electrospray ionization mass spectrometry: an essential tool for the characterization of complex glycoprotein digests

The B-domain of recombinant human Factor VIII comprises 909 amino acids and is extensively N- and O-glycosylated, in that at least 20 different sites are occupied by numerous carbohydrate structures. This domain was incubated with trypsin and subjected to liquid chromatography electrospray ionization mass spectrometry analysis, using an electrospray orthogonal acceleration time-of-flight mass spectrometer as the detector for a capillary reversed phase HPLC separation of the digest. The inherent high mass resolution afforded by this instrument provides both ion charge state determination and high accuracy mass measurement that are of significant advantage in defining such highly complex mixtures.     

On-line coupling of micellar electrokinetic chromatography to electrospray mass spectrometry

The possibility of selectivity enhancement in capillary electrophoresis-mass spectrometry (CE-MS) by hyphenating micellar electrokinetic chromatography (MEKC) and electrospray mass spectrometry (MS) is described for two quaternary ammonium compounds. Direct coupling of MEKC to MS is hazardous because of the contamination of the ion source due to presence of an excess of micelle forming agent in the MEKC buffer. Therefore, a coupled-capillary setup with the possibilities of voltage switching and buffer renewal has been designed. Such a system allows on-line heartcutting of the zones of interest in the MEKC capillary with subsequent transfer via a second capillary to the mass spectrometer.     

Characterization of carbohydrates using a combination of derivatization, high-performance liquid chromatography and mass spectrometry

Mice trisomic for the distal portion of MMU 16 (Ts65Dn) were examined for differences in jejunal function and plasma amino acids as compared to diploid controls. Eighteen control and 19 Ts65Dn mice were compared for whole-body and intestinal O2 consumption, jejunal glucose uptake, and plasma amino acid concentrations. Ts65Dn mice consumed less (P < 0.02) O2 per gram of fasted body weight. No significant differences were found in either active or passive glucose uptake. Oxygen consumption by jejunal tissue was not different between Ts65Dn and control mice. The apparent energetic efficiency of jejunal active glucose uptake (eta mol ATP expended/eta mol glucose uptake) was significantly higher (115.6 vs. 80.8; P < 0.05) in Ts65Dn mice. Histomorphometric analysis of jejunal mucosa showed that Ts65Dn mice had shorter villus height (P < 0.04) and decreased planar villus circumference (P = 0.05). No differences were found in total jejunal protein (microgram/g) or DNA (mg/g) concentrations. Significantly higher concentrations of plasma tyrosine, phenylalanine, valine, leucine, isoleucine, and citrulline (P < 0.05) were found in Ts65Dn mice. Lower plasma concentrations of hydroxyproline were detected in Ts65Dn mice (P < 0.05). These data suggest that Ts65Dn mice have anomalies in digestive function and amino acid metabolism as compared to normal, diploid controls.     

High-performance liquid chromatography with electrospray ionization mass spectrometry and diode array ultraviolet detection in the identification of flavonol aglycones and glycosides in berries

High-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) was used to study flavonol aglycones and glycosides in berries. For the identification of aglycones, photodiode-array detection (DAD) was also used. The HPLC-ESI-MS technique is highly valuable in the identification of flavonol aglycones and glycosides from berry extracts. This ionization technique provides information on the structure of the aglycones and glycosides without time-consuming pre-purification or derivatization steps. Quercetin aglycone was identified with both ESI-MS and DAD in all of the berries studied. Myricetin aglycone was identified with both techniques in three berries. Hexose, deoxyhexose-hexose and pentose derivatives of quercetin were the most abundant flavonol glycosides identified. Two glycosides of myricetin and one glycoside of kaempferol were identified in blackcurrant. To confirm the data obtained using the HPLC-ESI-MS procedure, fractions of the glycosides from four berries were separated, hydrolyzed, silylated and the sugars were analyzed using gas chromatography-mass spectrometry.     

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.     

Identification of eicosanoids in the red algae, Gracilaria asiatica, using high-performance liquid chromatography and electrospray ionization mass spectrometry

The "gold standard" inflow cytometric DNA analysis of breast cancer uses fresh tumor cells simultaneously labeled for cytokeratin (CK) and DNA. We developed a 2-parameter CK-DNA flow assay suitable for archival, paraffin-embedded tissue (PT). Six anti-CK monoclonal antibodies were tested by immunocytochemistry and our assay for staining of nuclei extracted from PT breast cancers by combination pepsin-trypsin digestion. Clone CAM 5.2 was inadequate for PT nuclear suspensions, but a cocktail of 2 anti-CK clones (AE1/AE3 and KL-1) distinguished epithelial from nonepithelial nuclei in 2-parameter flow dot plots. We studied 82 routine PT breast tumors by our assay and used a univariate flow DNA histogram based on fresh biopsy tissue for comparison. Three histogram data quality indicators were improved. A trend toward higher S-phase fractions was found for DNA diploid PT tumors, although when inflammation was evident histologically, the increment in S-phase fraction with gating was often marked. CK gating identified PT tumors containing concurrent CK-positive DNA diploid and nondiploid populations (27 of 56 DNA nondiploid histograms). By excluding nonepithelial nuclei, 2-color CK-DNA flow methods may increase the accuracy of ploidy and S-phase fraction measurements. Our method appears superior to previous techniques using clone CAM 5.2 for labeling of archival breast cancers.     

A microscale electrospray interface incorporating a monolithic, poly(styrene-divinylbenzene) support for on-line liquid chromatography/tandem mass spectrometry analysis of peptides and proteins

A methodology is described for creating a monolithic chromatography support within a pulled fused-silica electrospray needle. The monolith was formed from a mixture of styrene, divinylbenzene, 1-dodecanol, and toluene using 2,2'-azobis(isobutyronitrile) as the catalyst. The mixture was loaded into 150-micron-i.d. fused-silica capillary tubing with a pulled 5-10-micron needle tip at one end. Polymerization at 65 degrees C followed by removal of the porogen material yielded a stable, porous, monolithic support which had excellent properties for the separation and on-line, electrospray, mass spectrometry analysis of peptides and proteins. The performance of the monolith-filled electrospray needles was compared with similar needles filled with commercial C18 silica and polymeric particulate supports. Separation efficiencies for both protein and peptide mixtures were generally equal to or better than the particulate supports at comparable pressures and flow rates. The ion chromatograms derived from the on-line MS analysis were remarkably free from chemical background signals that often complicate the LC/MS analysis of femtomole amounts of sample. Good sequence coverage was obtained by LC/MS/MS analysis of the peptide mixture obtained from a protein isolated by silver-stained gel electrophoresis. The capability of the monolith to do peak parking experiments was demonstrated by the characterization of an immunoreactive HPLC fraction. The simple fabrication method, chromatographic performance, and robust nature of these microscale integrated column electrospray sources make them ideally suited for high-sensitivity tandem LC/MS analyses.     

Negative ion electrospray ionization tandem mass spectrometry in the structural characterization of penicillins

The mass spectrometry (MS) behaviour of ten commercially available penicillins has been studied by means of electrospray and multiple-stage MS/MS experiments performed using an ion trap instrument. For all the examined compounds negative ions are produced under ESI conditions, with a yield two or three orders of magnitude higher than that observed for positive ions. MSn experiments indicate the occurrence of a fragmentation pathway related to the beta-lactam ring, different from that usually described for positive ions of these compounds, and provide structural information on both the beta-lactam ring and the side chains.     

Mapping of phosphorylation sites of gel-isolated proteins by nanoelectrospray tandem mass spectrometry: potentials and limitations

Precursor ion scans have proven to be extremely useful for the characterization of unseparated peptide mixtures. In conjunction with the nanoelectrospray source, precursor ion scans provide a sensitive tool for the detection of posttranslationally modified peptides and have been used to determine phosphorylation sites of proteins digested in solution. In this report, we extend our previous work to the determination of protein phosphorylation sites of gel-isolated proteins. The in-gel digestion procedure developed in our laboratory for protein microsequencing was found to be suitable for phosphorylation mapping as well. The risk of losing hydrophilic peptides in the desalting step was decreased by using column packing material designed for the purification of oligonucleotides and by adjusting the pH conditions to the needs of phosphopeptide analysis. With this method, the tryptic phosphopeptides of beta-casein were detected after in-gel digestion at a sensitivity of 250 fmol of protein applied to the gel. The phosphorylation sites of two other proteins, Src-delta U and Op18, have similarly been mapped. Subpicomole to low-picomole amounts of protein starting material are needed in general, although we and others have reported attomole sensitivity for the detection of model phosphopeptides using precursor ion scans. This indicates that the success in determining phosphorylation sites depends crucially on the digestion, extraction, and detection efficiency for individual phosphopeptides.     

High performance liquid chromatography mobile phase composition optimization for the quantitative determination of a carboxylic acid compound in human plasma by negative ion electrospray high performance liquid chromatography tandem mass spectrometry

A systematic investigation was undertaken to study the effects of varying concentrations of additives in the acetonitrile/water high performance liquid chromatography mobile phase, especially formic acid and ammonium formate, on the negative ion electrospray response of a carboxylic acid compound. The study showed that the response progressively decreased with increase in the formic acid concentration. While such a decrease in the response could be qualitatively explained by the decrease in the concentration of the ionized form of the carboxylic acid compound due to the lower pH of the mobile phase, the change in response was not as large as expected from the change of the concentration of the ionized form. The response also progressively decreased with increase in the ammonium formate concentration but the decrease cannot be explained by the change in the pH of the mobile phase. Although the best negative ion electrospray response was obtained with a water/acetonitrile mobile phase that contained no additives at all, the retention time of the analyte was not found to be adequately reproducible on repeated injections. Thus, this mobile phase was deemed unacceptable for practical, routine use. Comparing formic acid against ammonium formate, the former was preferable since it caused a smaller attenuation of the negative ion response. Equally important was the fact that addition of formic acid had the desirable effect of maintaining a reasonably high capacity factor (k') for the analyte even at a relatively high acetonitrile concentration. A concentration of 1 mM formic acid in the mobile phase was large enough to achieve the reproducible elongated retention time for the analyte, with a loss in the analyte response of about 60% only. It should be noted that the sensitivity achieved with the 1 mM formic acid mobile phase, in which the carboxylic acid is expected to be about 10% in the ionized form, is about 9 times better than the sensitivity achieved in the 1 mM ammonium formate mobile phase, in which the carboxylic acid is expected to be about 99% in the ionized form.