Elucidation of peptide metabolism by on-line immunoaffinity liquid chromatography mass spectrometry

An immunoaffinity chromatography extraction capillary liquid chromatography separation has been coupled to electrospray ionization mass spectrometry for on-line characterization of drug metabolites of a therapeutic peptide in plasma. It is demonstrated that the selectivity, sensitivity and molecular weight data provided by immunoaffinity chromatography coupled to liquid chromatography/mass spectrometry provides a means of rapidly achieving qualitative determinations of small amounts of material in complicated biological matrices such as plasma. The ability to detect the peptide in rat plasma at a level of 10 ng/mL is demonstrated using this method. In addition, experiments to study the epitope of the peptide by enzymatic digestion and mass spectrometry are also discussed. The method is proposed as an alternative approach to studying the metabolism of therapeutic peptides.     

Application of gas and liquid chromatography coupled to time‐of‐flight mass spectrometry in pesticides: Multiresidue analysis

Analysis of pesticide residues in water and food matrices is an active research area closely related to food safety and environmental issues. In this aspect mass spectrometry (MS) coupled to gas chromatography (GC) and liquid chromatography (LC) has been increasingly used in the analysis of pesticide residues in water and food. The increasing interest in application of high‐resolution mass spectrometry with time‐of‐flight (TOF) and hybrid triple quadrupole TOF in pesticide analysis is due to its capability of performing both targeted and nontargeted analysis. This article discusses an overview of the application of GC‐TOF‐MS and LC‐TOF‐MS in water and food matrices.     

Qualitative and quantitative analysis of low molecular weight compounds by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrices

A major problem hampering the use of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for quantitative measurements is the inhomogeneous distribution of analytes and matrices in solid sample preparations. The use of ionic liquids as matrices for the qualitative and quantitative analysis of low molecular weight compounds like amino acids, sugars and vitamins was investigated. The ionic liquid matrices are composed of equimolar combinations of classical MALDI matrices (sinapinic acid, alpha-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid) with organic bases. These matrix systems allow a homogenous sample preparation with a thin ionic liquid layer having negligible vapour pressure. This leads to a facilitated qualitative and quantitative measurement of the analytes compared with classical solid matrices.     

Recent progress in the analysis of sugar monomers from complex matrices using chromatography in conjunction with mass spectrometry or stand-alone tandem mass spectrometry

Analysis of trace levels of carbohydrate monomers in complex matrices requires excellent discrimination of the peaks of interest from background noise. Minimizing contaminating peaks introduced during sample preparation and chromatography is extremely important. However, the exquisite selectivity of the mass spectrometer is essential as a chromatographic detector in this regard. Traditionally gas chromatography-mass spectrometry (GC-MS) has been the method of choice for trace analysis of derivatized carbohydrates. Recent improvements in commercial tandem mass spectrometers (MS-MS) are encouraging the use of GC-MS-MS for improved specificity in trace analysis. There has also been an explosion in applications of electrospray ionization (ESI) for sensitive introduction of polar molecules (including sugars) into the mass spectrometer. This has encouraged ongoing developments in high-performance liquid chromatography-mass spectrometry (LC-MS) and MS-MS of underivatized carbohydrates. This has the potential to dramatically simplify sample preparation. However, as yet LC-MS and MS-MS do not match the sensitivity of GC-MS or GC-MS-MS. Developments in analysis of sugar monomers from complex matrices using chromatography (GC/LC) in conjunction with mass spectrometry (MS, MS-MS) or stand-alone MS-MS are discussed.     

High‐efficiency sample preparation approach to determine acrylamide levels in high‐fat foods

An improved sample preparation method was developed to enhance acrylamide recovery in high‐fat foods. Prior to concentration, distilled deionized water was added to protect acrylamide from degradation, resulting in a higher acrylamide recovery rate from fried potato chips. A Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) was used for the first time to analyze acrylamide levels using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry, displaying good separation of acrylamide from interference. A solid‐phase extraction procedure was avoided, and an average recovery of >89.00% was achieved from different food matrices for three different acrylamide spiking levels. Good reproducibility was observed, with an intraday relative standard deviation of 0.04–2.38%, and an interday relative standard deviation of 2.34–3.26%. Thus, combining the improved sample preparation method for acrylamide analysis with the separation on a Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry is highly useful for analyzing acrylamide levels in complex food matrices.     

Determination of alkylphenol and alkylphenolethoxylates in biota by liquid chromatography with detection by tandem mass spectrometry and fluorescence spectroscopy

A quantitative method for the simultaneous determination of octylphenol, nonylphenol and the corresponding ethoxylates (1 to 5) in biota is presented. Extraction methods were developed for egg and fish matrices based on accelerated solvent extraction followed by a solid-phase extraction cleanup, using octadecylsilica or aminopropyl cartridges. Identification and quantitation were accomplished by liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) and compared to the traditional liquid chromatography with fluorescence spectroscopy detection. LC-MS-MS provides high sensitivity and specificity required for these complex matrices and an accurate quantitation with the use of 13C-labeled internal standards. Quantitation limits by LC-MS-MS ranged from 4 to 12 ng/g in eggs, and from 6 to 22 ng/g in fish samples. These methods were successfully applied to osprey eggs from the Chesapeake Bay and fish from the Great Lakes area. Total levels found in osprey egg samples were up to 18 ng/g wet mass and as high as 8.2 microg/g wet mass in the fish samples.     

Determination of paramethoxyamphetamine and other amphetamine-related designer drugs by liquid chromatography/sonic spray ionization mass spectrometry

Paramethoxyamphetamine (PMA) is an amphetamine-like designer drug that has emerged recently on the European illicit drug market. This drug has a wicked reputation, as a number of lethal intoxications have occurred. A method using high-performance liquid chromatography coupled to ion trap based mass spectrometry (LC/MS) is described for the determination of this compound together with 3,4-methylenedioxymethamphetamine (XTC or MDMA), amphetamine and 3,4-methylenedioxyamphetamine (MDA) in human matrices. A liquid/liquid extraction (LLE) was applied to whole blood, urine and postmortem tissues. Reversed-phase liquid chromatography was performed on a narrow-bore phenyl-type column at a flow rate of 0.3 mL/min. A switch box allowed disposal of early-eluting irrelevant material to waste, protecting the mass spectrometer from contamination. The column effluent was directed into an ion trap mass spectrometer by a sonic spray ionization (SSI) interface. The method was validated for all three matrices, proving the applicability of SSI even when dealing with complex biological matrices. The within-and between-day precisions were less than 17.5% and accuracy was below 16.2%. Weighted (1/x) quadratic calibration curves were generated ranging from 10 to 1000 ng/mL (blood and urine) or 20 to 2000 ng/g (tissue) and correlation coefficients (r(2)) always exceeded 0.995. In addition, the mass spectrum of PMA is given together with a proposed fragmentation pattern for the obtained LC/MS spectrum. This information can be useful for future identification of PMA with LC/MS in biological matrices as well as in confiscated powders or tablets.     

Characterization of beam-induced reactions occurring in liquid secondary ion mass spectrometry/fast-atom bombardment by tandem mass spectrometry

Department of General and Physical Chemistry, University of Liege, Liege, Belgium A specific beam-induced secondary reaction involving the condensation of hydroxylic matrices with some organic groups (aldehydes, ketones, etc.) accompanied by the loss of a water molecule was investigated by liquid secondary ion mass spectrometry/fast-atom bombardment (LSIMS/FAB). A mechanistic scheme and a structure of the induced product are proposed. The features of this secondary reaction were studied and the influence of the types of solutes, acidic additives, and matrices analyzed. Rather than a drawback, LSIMS/FAB mass spectrometry can take advantage of this matrix effect to infer analytical information through tandem mass spectrometry experiments. Specific neutral loss scans can be conducted to highlight beam-induced reactive molecules, even when the detection of these species is prevented in normal scan spectra by other surface-active components.     

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography–triple quadrupole mass spectrometry‐based quantitative protein assays use bottom‐up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top‐down reversed‐phase liquid chromatography–triple quadrupole mass spectrometry method for analysis of cancer‐related intact proteins was evaluated. Seven growth factors (5.5–26.5 kDa; isoelectric points: 4.6–9.9) were analyzed on a wide‐pore C4 column. The optimized method was performed at 30°C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1–500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields.     

Selective enrichment and quantification of okadaic acid in shellfish using an immunomagnetic‐bead‐based liquid chromatography with tandem mass spectrometry assay

Okadaic acid is a marine biotoxin that primarily occurs in shellfish and can cause diarrheic shellfish poisoning in humans. When analyzing biological samples using liquid chromatography with tandem mass spectrometry, the presence of complex matrices is a major issue. Thus, it is crucial to selectively and simply extract the target analyte from samples and minimize matrix effects simultaneously. To meet this need, an immunomagnetic‐bead‐based liquid chromatography with tandem mass spectrometry method was developed to detect okadaic acid in shellfish. Magnetic beads bound to monoclonal antibody against okadaic acid were used as affinity probes to specifically enrich okadaic acid in samples, which effectively eliminated matrix effects. A magnetic separator was used to aggregate and separate magnetic particles from sample matrices, and methanol was used to elute okadaic acid from the magnetic beads. Standard solution prepared with methanol was employed directly for quantitative analysis. Several experimental conditions were optimized to improve performance. The method is of interest as a rapid (10 min) sample clean‐up and selective enrichment tool, and it showed good linearity and sensitivity, with reported limits of detection and quantitation of 3 and 10 μg/kg, respectively. Fifty‐three shellfish samples from an aquatic products market were tested using this method, and four samples positive for okadaic acid were found.     

Characterization of the molecular mass distribution of pullulans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using 2,5-dihydroxybenzoic acid butylamine (DHBB) as liquid matrix

The performances of several matrices were investigated for the accurate determination of the molecular mass distributions of pullulans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The ionic liquid matrix (ILM) 2,5-dihydroxybenzoic acid butylamine (DHBB) gave better and more reliable results than the crystalline matrices 2,5-dihydroxybenzoic acid (DHB) and 2,4,6-trihydroxyacetophenone (THAP). With the ILM it was possible to obtain spectra of pullulans up to more than 100 kDa, the highest molar mass reported thus far. Owing to the known advantages of liquid matrices providing better spot-to-spot reproducibility, an almost noise-free spectrum and constant baselines were obtained when working under optimized conditions. In particular, the extent of in-source fragmentation occurring with this group of fragile polymers was considerably and decisively reduced. Thus, a more reliable representation of the true oligomer and polymer distributions is experimentally attainable, especially for distributions with small polydispersity values. The maximum error in the measured distribution associated with fragmentation was estimated by model calculations describing the changes in the polymer distribution upon different probabilities of fragmentation events. These simulation results indicated that the data obtained by MALDI-TOFMS using the liquid DHBB matrix were of high reliability. In particular, the average value of the distributions, M(w), and the polydispersity were obtained with predicted uncertainties of between 3 and 15% depending on the width of the distribution and the mass of the polymers. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Mass defect profiles of biological matrices and the general applicability of mass defect filtering for metabolite detection

Recent examples have demonstrated that the high-resolution liquid chromatography/mass spectrometry (LC/MS)-based mass defect filtering (MDF) technique was effective in selectively detecting drug metabolites regardless of their molecular weights or fragmentation patterns. The main objective of the current study was to evaluate the general applicability of MDF for drug metabolite detection in typical biological matrices. Mass defect profiles of commonly used biological matrices including plasma, urine, bile, and feces were obtained using an LTQ FT mass spectrometer and were compared with those of 115 commonly prescribed drugs. The mass defect profiles were presented as two-dimensional Y-X plots with the determined mass defects of components on the y-axis versus the corresponding m/z values on the x-axis. The mass defect profiles of the matrices appeared to be similar for each type of matrix across species, yet marked differences were apparent between matrices of a given species. The mass defect profiles of components in plasma, bile, and feces showed significant separation from most of the 115 drugs. The mass defect profiles of urine did not show such clean separation from that of the 115 drugs. The results suggest that MDF has a broad applicability for selective detection of drug metabolites in plasma, bile and feces although the selectivity for detecting urinary drug metabolites is not as good as in the other matrices. In addition, the mass defect profiles of the biological matrices allow for prediction of the effectiveness of MDF for certain applications, and for designing specific MDF windows for selective detection of drug metabolites.     

Novel, highly robust method of carbohydrate pre-purification by two-dimensional liquid chromatography prior to liquid chromatography/mass spectrometry or gas chromatography/mass spectrometry

We describe a novel two-dimensional liquid chromatography (2D-LC) method for fast and robust isolation and concentration of low abundant carbohydrates (sorbitol, glycerol) from biological matrices (plasma and urine). Off-line pre-purified fractions, enriched by analyte of interest, were analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS-MS). Initial 2D-LC automated sample pre-purification improved MS detection, eliminated matrix effects, and achieved high sensitivity (picogram detection limit) with a 6 min runtime and increased column lifetime. Using this method we have analyzed more than 1300 samples from biological matrices without column replacement.     

Determination of perfluorochemicals in biological, environmental and food samples by an automated on-line solid phase extraction ultra high performance liquid chromatography tandem mass spectrometry method

A rapid on-line solid phase extraction ultra high performance liquid chromatography tandem mass spectrometry method was developed for the identification and quantitation of nine perfluorinated compounds in matrices of environmental, biological and food interest. Pre-treatment, solid phase extraction, chromatographic and mass detection conditions were optimised, in order to apply the whole methodology to the analysis of different matrices. Particular attention was devoted to the evaluation of matrix effect and the correlated phenomena of ion enhancement or suppression in mass spectrometry detection. LOD and LOQ range from 3 to 15ngL(-1) and from 10 to 50ngL(-1), respectively. Method detection limits (MDLs) were also calculated for each kind of matrix. The recovery, evaluated for each analyte, does not depend on analyte concentration in the explored concentration range: average Rˉ% values are always greater than 82.9%. In the whole, the results obtained for samples of river waters, blood serum, blood plasma, and fish confirm the ubiquitous presence of perfluorinated compounds, as recently denounced by many sources.     

Fragmentation pathways of heroin-related alkaloids revealed by ion trap and quadrupole time-of-flight tandem mass spectrometry

The electrospray ionization (ESI) ion trap and quadrupole time-of-flight (QqToF) mass spectra of heroin and seven related alkaloids, i.e., morphine, codeine, O-6-monoacetylmorphine (6-MAM), thebaine, acetylcodeine, papaverine and narcotine, have been extensively investigated in this work. The ESI mass spectrometric fragmentation pathways of protonated 6-MAM, heroin, acetylcodeine, and thebaine were comprehensively elucidated for the first time with the aid of high-resolution mass spectrometry. It was found that cleavage of the piperidine ring was the featured fragmentation route of six of the compounds, although not of papaverine and narcotine. In addition, a simple high-performance liquid chromatography (HPLC)-based separation method gave baseline resolution of all eight components. This study could play an important role in the screening for these alkaloids in different matrices by HPLC coupled to tandem mass spectrometry (MS/MS).     

Ionic liquids as matrices in microfluidic sample deposition for high-mass matrix- assisted laser desorption/ionization mass spectrometry

Sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) via a microfluidic deposition device using ionic liquid matrices addresses several problems of standard protocols with crystalline matrices, such as the heterogeneity of sample spots due to the co-crystallization of sample and matrix and the limited capability for high-throughput analysis. Since ionic liquid matrices do not solidify during the measurement, the resulting sample spots are homogeneous. The use of these matrices is also beneficial for automated sample preparation, since crystallization of the matrix is avoided and, thus, no clogging of the spotting device can occur. The applicability of ionic liquids to the analysis of biomolecules with high molecular weights, up to ≈ 1 MDa is shown, as well as a good sensitivity (5 fmol) for recombinant human fibronectin, a protein with a molecular weight of 226 kDa. Microfluidic sample deposition of proteins with high molecular weights will, in the future, allow parallel sample preparation for MALDI-MS and for electron microscopy.     

Observation of apigenin anionic clusters in the gas phase

The ubiquity and favorable medicinal properties of flavonoids make essential the determination of flavonoid levels in various matrices. While developing a liquid chromatography/tandem mass spectrometry method for the analysis of the flavonoid, apigenin, anionic oligomers and nitrate- and chloride-bound clusters of this compound were observed. Tandem mass spectrometry of these oligomers and cluster ions showed the cleavage of apigenin molecules from the precursor. The observation of these cluster ions shows the possibility of post-column derivatization techniques to enhance specificity in analysis. Copyright 2000 John Wiley & Sons, Ltd.     

UV-laser ablation of ionic liquid matrices

Ionic liquid matrices are a new class of matrices used in MALDI mass spectrometry. The ablation process of several ionic liquid matrices was studied by determining the velocity distribution of ablated neutral matrix molecules. This was done by a postionization approach, where the neutrals were ionized in the ablation plume by a second laser pulse. It was found that a second, time-delayed ablation event occurs consisting completely of neutral molecules. To explain this, the reflected-shockwave model is used, which assumes that the shockwave emerging from the laser ablation is reflected at the sample holder surface. When the shockwave arrives at the sample surface it causes a second ablation.     

Simultaneous determination of phenolic compounds in Equisetum palustre L. by ultra high performance liquid chromatography with tandem mass spectrometry combined with matrix solid‐phase dispersion extraction

A method based on matrix solid‐phase dispersion extraction followed by ultra high performance liquid chromatography with tandem mass spectrometry is presented for the extraction and determination of phenolic compounds in Equisetum palustre. This method combines the high efficiency of matrix solid‐phase dispersion extraction and the rapidity, sensitivity, and accuracy of ultra high performance liquid chromatography with tandem mass spectrometry. The influential parameters of the matrix solid‐phase dispersion extraction were investigated and optimized. The optimized conditions were as follows: silica gel was selected as dispersing sorbent, the ratio of silica gel to sample was selected to be 2:1 (400/200 mg), and 8 mL of 80% methanol was used as elution solvent. Furthermore, a fast and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the determination of nine phenolic compounds in E. palustre. This method was carried out within <6 min, and exhibited satisfactory linearity, precision, and recovery. Compared with ultrasound‐assisted extraction, the proposed matrix solid‐phase dispersion procedure possessed higher extraction efficiency, and was more convenient and time saving with reduced requirements on sample and solvent amounts. All these results suggest that the developed method represents an excellent alternative for the extraction and determination of active components in plant matrices.     

Review of the QuEChERS method for the analysis of organic pollutants: Persistent organic pollutants,polycyclic aromatic hydrocarbons,and pharmaceuticals

In multi-residue pesticide analysis, the quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method has replaced less efficient traditional extraction methods due to its many advantages. In addition to pesticide analysis, this method has been widely used for the detection and analysis of pharmaceuticals, polycyclic aromatic hydrocarbons (PAHs), and several persistent organic pollutants (POPs), including dioxins, polychlorinated biphenyls, perfluoroalkyl substances, and brominated flame retardants in food, biological, and environmental matrices. The analysis of PAHs and POPs is challenging due to the properties of the target compounds and their low concentrations in complex matrices. This review summarizes previously reported the QuEChERS extraction approaches to the analysis of a wide range of analytes. The QuEChERS approaches, which include dispersive solid phase extraction (d-SPE), have generally been combined with either gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) analysis. Further on, in recent years, GC and LC-tandem mass spectrometry has been utilized with the QuEChERS extraction due to its high selectivity, sensitivity, and specificity. This enables the extraction methods for target analytes to be modified through the selection of different extraction solvents, salt formulations, and buffers for salting-out partitioning and the selection of different d-SPE and SPE sorbents for the clean-up process. The most significant advantage of this method is that concentration steps are not required. This review aims to provide an up-to-date overview of information regarding the modification of extraction techniques based on target compounds and sample matrices.