Comprehensive gas chromatography-electron ionisation mass spectrometric analysis of fatty acids and sterols using sequential one-pot silylation: quantification and isotopologue analysis.

RATIONALE: Fatty acids and sterol lipids play crucial roles in several biological processes and several biological facts underline the interconnection between these lipid classes. Therefore, it is of interest to develop a comprehensive method analysing both classes in the form of their most favourable derivatives suitable for quantification and isotopologue analysis. METHODS: Lipids were derivatised by a sequential one-pot procedure using N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MtBSTFA) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). No clean-up or concentration steps were necessary. The prepared samples were directly available for gas chromatography-electron ionisation mass spectrometric (GC-EI-MS) analysis on a standard column. For quantification, the SIM mode was used and for isotopologue analysis scheduled scan mode was applied. RESULTS: Development of a sequential one-pot derivatisation for GC-EI-MS allowing comprehensive analysis of fatty acids and sterols as their most favourable derivatives. Validation carried out using human plasma, comparison with certified NIST plasma. LLOQ of usually 3.3 ng/mL achieved. Isotopologue analysis of 2-[(13)C]-acetate incorporation in HL-60 cells proving feasibility of method. CONCLUSIONS: The presented method successfully combines two consecutive silylation reactions in one pot, enabling the analysis of both fatty acids and sterols in a comprehensive analytical method. The method has great potential for the quantification of lipids as well as the comprehensive study of both biochemical pathways, using [(13)C]-flux analysis.

EC-SPE-stripline-NMR analysis of reactive products: a feasibility study.

Flow-through electrochemical conversion (EC) of drug-like molecules was hyphenated to miniaturized nuclear magnetic resonance spectroscopy (NMR) via on-line solid-phase extraction (SPE). After EC of the prominent p38α mitogen-activated protein kinase inhibitor BIRB796 into its reactive products, the SPE step provided preconcentration of the EC products and solvent exchange for NMR analysis. The acquisition of NMR spectra of the mass-limited samples was achieved in a stripline probe with a detection volume of 150 nL offering superior mass sensitivity. This hyphenated EC-SPE-stripline-NMR setup enabled the detection of the reactive products using only minute amounts of substrate. Furthermore, the integration of conversion and detection into one flow setup counteracts incorrect assessments caused by the degradation of reactive products. However, apparent interferences of the NMR magnetic field with the EC, leading to a low product yield, so far demanded relatively long signal averaging. A critical assessment of what is and what is not (yet) possible with this approach is presented, for example in terms of structure elucidation and the estimation of concentrations. Additionally, promising routes for further improvement of EC-SPE-stripline-NMR are discussed.

Recent Advancements in the LC- and GC-Based Analysis of Malondialdehyde (MDA): A Brief Overview.

Malondialdehyde (MDA) is an end-product of lipid peroxidation and a side product of thromboxane A(2) synthesis. Moreover, it is not only a frequently measured biomarker of oxidative stress, but its high reactivity and toxicity underline the fact that this molecule is more than "just" a biomarker. Additionally, MDA was proven to be a mutagenic substance. Having said this, it is evident that there is a major interest in the highly selective and sensitive analysis of this molecule in various matrices. In this review, we will provide a brief overview of the most recent developments and techniques for the liquid chromatography (LC) and gas chromatography (GC)-based analysis of MDA in different matrices. While the 2-thiobarbituric acid assay still is the most prominent methodology for determining MDA, several advanced techniques have evolved, including GC-MS(MS), LC-MS(MS) as well as several derivatization-based strategies.

Nanofractionation spotter technology for rapid contactless and high-resolution deposition of LC eluent for further off-line analysis.

The development of a contactless postcolumn spotter technology capable of rapidly and accurately depositing LC eluent onto another platform (e.g., 1536-well microtiter plates) is described. Many detection methodologies are suitable for online analysis, such as mass spectrometry, UV-vis, and fluorescence. In some cases, when online analysis is less suitable, off-line postcolumn analysis is the methodology of choice and usually relies on LC-based fractionation prior to detection (e.g., MALDI-MS, Raman spectrsocopy, biochemical assays). As fractionation generally involves loss in resolution, the technology described here allows high-resolution contactless fractionation by tailoring the fractionation frequency to the chromatographic peaks and mixing in of postcolumn reagents. Droplet ejection at frequencies of at least 6 Hz could be performed in the nanoliter to low microliter range with repeatabilities of ∼6%. Furthermore, multiple droplets can be ejected at the same position thereby allowing adjustment of fractionation volume and speed. The technology was evaluated, optimized, and validated prior to two proof-of-principle demonstrations comprising off-line chemical detection of injected fluorescein and off-line postcolumn biochemical detection of acetylcholine-binding protein ligands, both based on 1536-well plate reader analysis.

Mild and selective labeling of malondialdehyde with 2-aminoacridone: assessment of urinary malondialdehyde levels.

Malondialdehyde (MDA) has become a well-established biomarker for oxidative stress. The most commonly used way to determine urinary MDA levels is the thiobarbituric acid (TBA) assay, which suffers from several drawbacks. In this manuscript, we describe a novel derivatization strategy for the highly sensitive and selective fluorescence-based determination of MDA in urinary samples. The methodology is based on the mild labeling of MDA with 2-aminoacridone, which can be carried out in aqueous citrate buffer at 40 °C, yielding a highly fluorescent substance. No further sample preparation than mixing with the necessary chemicals is necessary. The formed MDA derivative can conveniently be separated from the label itself and matrix constituents by gradient LC in less than 5 minutes on a cyano-based reversed-phase material. The method was validated with respect to matrix effects, linearity, selectivity and sensitivity (values as low as 1.8 nM for the LOD and 5.8 nM for the LOQ could be achieved). Standard addition quantitation was applied for the determination of MDA in human urine samples. Additionally, the protocol was applied to the measurement of a stability indicating analysis of MDA in urine at different storage conditions.

Analysis of acetylcholinesterase inhibitors: bioanalysis, degradation and metabolism.

Alzheimer's is a neurodegenerative disease. Its symptoms are attributed to a deficiency of cholinergic neurotransmission. The drugs of choice for the treatment of Alzheimer's disease are acetylcholinesterase (AChE) inhibitors. Starting in the 1980's from non-specific AChE inhibitors, the first-generation drugs such as physostigmine, a second generation of more selective and better tolerated products has been developed. Methods to detect and quantify these drugs and their metabolites in biological samples have been developed for analysis in plasma, blood, urine and cerebrospinal fluid. Diverse detection techniques have been used, such as ultraviolet, fluorescence, electrochemical and mass spectrometry. In this review, the methods applied to the analysis of these drugs and their metabolites in different biological matrices are reviewed and discussed. The stability of these drugs in biological matrices and under stress-conditions is also included in the discussion.

Structural elucidation of biologically active neomycin N-octyl derivatives in a regioisomeric mixture by means of liquid chromatography/ion trap time-of-flight mass spectrometry.

Structural elucidation of six regioisomers of mono-N-octyl derivatized neomycin is achieved using MS(n) (up to n = 4) on an ion trap time-of-flight (IT-TOF) instrument equipped with electrospray ionization. The mixture of six derivatized neomycin analogues was generated by reductive amination in a shotgun synthetic approach. In parallel to the liquid chromatography/mass spectrometry (LC/MS) detection, the antibacterial activity of the neomycin regioisomers was tested by post-column addition of buffer and bacterial inocula, subsequent microfractionation of the resulting mixture, incubation, and finally a chemiluminescence-based bioactivity measurement based on the production of bacterial ATP. The MS-based high-resolution screening approach described can be applied in medicinal chemistry to help in designing and producing new antibiotic substances, which is particularly challenging due to the high functionality of most antibiotic substances, therefore requiring advanced (hyphenated) separation and detection techniques for compound mixtures.

Targeted LC-MS derivatization for aldehydes and carboxylic acids with a new derivatization agent 4-APEBA.

Based on the template of a recently introduced derivatization reagent for aldehydes, 4-(2-(trimethylammonio)ethoxy)benzeneaminium dibromide (4-APC), a new derivatization agent was designed with additional features for the analysis and screening of biomarkers of lipid peroxidation. The new derivatization reagent, 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) contains a bromophenethyl group to incorporate an isotopic signature to the derivatives and to add additional fragmentation identifiers, collectively enhancing the abilities for detection and screening of unknown aldehydes. Derivatization can be achieved under mild conditions (pH 5.7, 10 degrees C). By changing the secondary reagent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of sodium cyanoborohydride), 4-APEBA is also applicable to the selective derivatization of carboxylic acids. Synthesis of the new label, exploration of the derivatization conditions, characterization of the fragmentation of the aldehyde and carboxylic acid derivatives in MS/MS, and preliminary applications of the labeling strategy for the analysis of aldehydes in urine and plasma are described.

Development of a microfluidic confocal fluorescence detection system for the hyphenation of nano-LC to on-line biochemical assays.

One way to profile complex mixtures for receptor affinity is to couple liquid chromatography (LC) on-line to biochemical detection (BCD). A drawback of this hyphenated screening approach is the relatively high consumption of sample, receptor protein and (fluorescently labeled) tracer ligand. Here, we worked toward minimization of sample and reagent consumption, by coupling nano-LC on-line to a light-emitting diode (LED) based capillary confocal fluorescence detection system capable of on-line BCD with low-flow rates. In this fluorescence detection system, a capillary with an extended light path (bubble cell) was used as a detection cell in order to enhance sensitivity. The technology was applied to a fluorescent enhancement bioassay for the acetylcholine binding protein, a structural analog of the extracellular ligand-binding domain of neuronal nicotinic acetylcholine receptors. In the miniaturized setup, the sensitive and low void volume LED-induced confocal fluorescence detection system operated in flow injection analysis mode allowing the measurement of IC(50) values, which were comparable with those measured by a conventional plate reader bioassay. The current setup uses 50 nL as injection volume with a carrier flow rate of 400 nL/min. Finally, coupling of the detection system to gradient reversed-phase nano-LC allowed analysis of mixtures in order to identify the bioactive compounds present by injecting 10 nL of each mixture.

Direct Dynamic Protein-Affinity Selection Mass-Spectrometry.

A new methodology is described enabling the affinity screening of potential ligands towards the human estrogen receptor alpha ligand binding domain (ERalpha-LBD). In-solution incubation is performed of the analyte and the His-tagged ERalpha-LBD. The bound complex is immobilized on a nickel-loaded protein-affinity selection column, where after the unbound fraction is removed. The immobilized protein-ligand complex is exposed to a decreased pH value and an increased organic modifier concentration releasing the ligand for MS detection, and precipitating the proteins on a filter positioned between the affinity column and the mass spectrometer. The trapping column can be regenerated for reuse at least 70 times. The advantages of the methodology over existing methodologies are the absence of a pre-concentration as well as a chromatographic separation step, resulting in a significantly shorter analysis time compared to previously described procedures, and in addition, allowing the determination of solutes with unfavorable chromatographic properties. The overall analysis time now can be reduced about 250% to approximately 6 min. Replacing the filters after every measurement results in an intra-day standard deviation of 14.8% and an inter-day standard deviation of 21.3%.

Identification of the Biotransformation Products of 2-Ethylhexyl 4-(N,N-Dimethylamino)benzoate.

Nowadays, 2-ethylhexyl 4-(N,N-dimethylamino)benzoate (EDP) is one of the most widely used UV filters in sunscreen cosmetics and other cosmetic products. However, undesirable processes such as percutaneous absorption and biological activity have been attributed to this compound. The in vitro metabolism of EDP was elucidated in the present work. First of all, the phase I biotransformation was studied in rat liver microsomes and two metabolites, N,N-dimethyl-p-aminobenzoic acid (DMP) and N-monomethyl-p-aminobenzoic acid (MMP), were identified by GC-MS analysis. Secondly, the phase II metabolism was investigated by means of LC-MS. The investigated reactions were acetylation and glucuronidation working with rat liver cytosol and with both human and rat liver microsomes, respectively. Analogue studies with p-aminobenzoic acid (PABA) were carried out in order to compare the well established metabolic pathway of PABA with the unknown biotransformation of EDP. In addition, a method for the determination of EDP and its two phase I metabolites in human urine was developed. The methodology requires a solid-phase extraction prior to LC-MS analysis. The method is based on standard addition quantification and has been fully validated. The repeatability of the method, expressed as relative standard deviation, was in the range 3.4-7.4% and the limit of detection for all quantified analytes was in the low ng mL(-1) range.

Microfractionation revisited: a 1536 well high resolution screening assay.

The aim of the here presented study was to combine high performance liquid chromatography with plate reader technology in order to overcome certain drawbacks of integrated online systems as well as offline plate reader approaches. The described method combines an "at-line" enzyme assay for the simultaneous bioactivity determination with parallel QTOF MS data acquisition for analyte identification. All biochemical reagents are added in an online mode directly to the column effluent (postcolumn addition/mixing), and the complete screening assay mixture is subsequently microfractionated into a 1536 well plate. The screening of a natural extract fortified with two well-known Protein Kinase A inhibitors and the identification of an inhibitor in a natural extract showed the applicability of the approach to detect bioactive compounds in low concentrations in a complex mixture. The described mode of operation utilizes today's plate reader technology to its full capacity and directly hyphenates it to a high resolution separation technique which has not been shown before. Furthermore, it allows coupling of a microbore HPLC with a biochemical screening assay without compromising resolution and overcomes problems associated with the 1536 well format.

Determination of Flavonoids and Resveratrol in Wine by Turbulent-Flow Chromatography-LC-MS.

Turbulent-flow chromatography (TFC) on-line coupled to liquid chromatography mass spectrometry (LC-MS) is used to determine flavonoids and resveratrol in different types of wines. A fully automated system was developed in which 10 mL of sample (diluted wine) was passed over a TFC column, after which the retained analytes were separated by reversed-phase LC and detected by negative ion mode atmospheric-pressure chemical ionization (APCI) MS. The method proved to be fast, non-laborious, robust and sensitive. The feasibility of the method was tested on several red, white and rose wines. Quantitation of resveratrol was possible using the standard addition procedure. Red wine showed the highest amount of resveratrol (4 mg L(-1)), while rose and white wine contained concentrations which were about ten fold lower.

Screening of protein-ligand interactions using dynamic protein-affinity chromatography solid-phase extraction-liquid chromatography-mass spectrometry.

A novel methodology is shown enabling the screening of mixtures of compounds for their affinity to a receptor protein. The system presented, dynamic protein-affinity chromatography solid-phase extraction (DPAC-SPE), overcomes the limitations of the existing methods by performing an incubation of the His-tagged protein with a mixture of possible ligands, which are still in their native conditions. This is followed by a fully automated affinity trapping step, coupled on-line to an LC-MS system in order to detect and identify the bound ligands. The system has been optimized using a commercially available on-line SPE system, using the estrogen receptor alpha (ERalpha) as model protein. A representative range of ligands with sub-nanomolar to millimolar affinities has been identified successfully from a mixture. The weakest binder that can be identified is norethindrone (approximately K(d)=0.1-1mM). The same setup also provides the possibilities to measure EC50 curves of both weak and strong binders.

Development and optimization of a system for comprehensive two-dimensional liquid chromatography with UV and mass spectrometric detection for the separation of complex samples by multi-step gradient elution.

Comprehensive two-dimensional liquid chromatography (LC x LC) is a powerful tool for the separation of complex biological samples. This technique offers the advantage of simplified automation and greater reproducibility in a shorter analysis time than off-line two-dimensional separation systems. In the present study, an LC x LC system is developed enabling simultaneous UV and MS detection, and which can be easily converted to a conventional reversed-phase LC-UV/MS system. In LC x LC, a 60-min reversed-phase LC separation with a linear solvent gradient in the first dimension is coupled to a second-dimension separation on a mixed-mode cation-exchange/reversed-phase column with a modulation time of 60s. The isocratic separation in the second-dimension column is optimized by the use of a multi-step gradient where the organic and the ionic modifier are varied independently. Intraday (n=3) and interday (n=4) variability of the retention times were evaluated with the complete system and found to be 0.5% and 0.7%, respectively. Good linearity was observed in calibration curves for three different compounds varying in polarity.

Automated detection of covalent adducts to human serum albumin by immunoaffinity chromatography, on-line solution phase digestion and liquid chromatography-mass spectrometry.

A generic method for the detection of covalent adducts to the cysteine-34 residue of human serum albumin (HSA) has been developed, based on an on-line combination of immunoaffinity chromatography for selective sample pre-treatment, solution phase digestion, liquid chromatography and tandem mass spectrometry. Selective anti-HSA antibodies immobilized on agarose were used for sample pre-concentration and purification of albumin from the chemically produced alkylated HSA. After elution, HSA and HSA adducts are mixed with pronase and directed to a reaction capillary kept at a digestion temperature of 70 degrees C. The digestion products were trapped on-line on a C18 SPE cartridge. The peptides were separated on a reversed-phase column using a gradient of organic modifier and subsequently detected using tandem mass spectrometry. Modified albumin samples consisted of synthetically alkylated HSA by the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine (NAPQI), and using the alkylating agent 1-chloro-2,4-dinitrobenzene (CDNB) as reference. The resulting mixture of alkylated versus non-modified albumin has been applied to the on-line system, and alkylation of HSA is revealed by the detection of the modified marker tetra-peptide glutamine-cysteine-proline-phenylalanine (QCPF) adducts NAPQI-QCPF and CDNB-QCPF. Detection of alkylated species was enabled by the use of data comparison algorithms to distinguish between unmodified and modified HSA samples. The in-solution digestion proved to be a useful tool for enabling fast (less than 2 min) and reproducible on-line digestion of HSA. A detection limit of 1.5 micromol/L of modified HSA could be obtained by applying 10 microL of NAPQI-HSA sample.

Selective quantitative bioanalysis of proteins in biological fluids by on-line immunoaffinity chromatography-protein digestion-liquid chromatography-mass spectrometry.

A quantitative method for the determination of proteins in complex biological matrices has been developed based on the selectivity of antibodies for sample purification followed by proteolytic digestion and quantitative mass spectrometry. An immunosorbent of polyclonal anti-bovine serum albumin (BSA) antibodies immobilized on CNBR agarose is used in the on-line mode for selective sample pretreatment. Next, the purified sample is trypsin digested to obtain protein specific peptide markers. Subsequent analysis of the peptide mixture using a desalination procedure and a separation step coupled, on-line to an ion-trap mass spectrometer, reveals that this method enables selective determination of proteins in biological matrices like diluted human plasma. This approach enhances substantially the selectivity compared to common quantitative analysis executed with immunoassays and colorimetry, fluorimetry or luminescence detection. Hyphenation of the immunoaffinity chromatography with on-line digestion and chromatography-mass spectrometry is performed and a completely on-line quantification of the model protein BSA in bovine and human urine was established. A detection limit of 170 nmol/l and a quantification limit of 280 nmol/l is obtained using 50 microl of either standard or spiked biological matrix. The model system allows fully automated absolute quantitative mass spectrometric analysis of intact proteins in biological matrices without time-consuming labeling procedures.

A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry.

The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.

On-line multidimensional liquid chromatography and capillary electrophoresis systems for peptides and proteins.

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated.

Extraction of isoflavone malonylglucosides from Trifolium pratense L.

Extraction of isoflavone malonylglucosides from red clover (Trifolium pratense L.) is a complicated procedure. This is due to the relatively unstable character of the thermolabile glucoside malonates as well as by action of native beta-glucosidases, resulting in a rapid degradation of malonylated glucosides into their corresponding aglucones. In this study, Tris was identified as a suitable beta-glucosidase inhibitor in red clover extracts, optimized at 350 mM Tris in 80% ethanol at pH 7.2. Extraction of fresh red clover leaves using Tris increased the concentration of malonate conjugated isoflavones approximately 13 to 24 times as opposed to extraction without Tris. A comparison of isoflavone profiles obtained after extraction with and without Tris of different plant organs of red clover and several species within the family Fabaceae suggests that the amount and/or activity of the degenerative beta-glucosidase enzymes vary for the different plant parts of red clover and among the species studied. Therefore, the use of standard extraction methods may well result in overestimation of the concentration of aglucones and consequently underestimation of the malonylglucoside isoflavones concentration depending on the plant species and plant part studied.