Quantitative analysis of N-acylphosphatidylethanolamine molecular species in rat brain using solid-phase extraction combined with reversed-phase chromatography and tandem mass spectrometry.

A novel method for the sensitive and selective identification and quantification of N-acylphosphatidylethanolamine molecular species was developed. Samples were prepared using a combination of liquid-liquid and solid-phase extraction, and intact N-acylphosphatidylethanolamine species were determined by reversed-phase high-performance liquid chromatography coupled to positive electrospray tandem mass spectrometry. As a result of their biological functions as precursors for N-acylethanolamines and as signaling molecules, tissue concentrations of N-acylphosphatidylethanolamines are very low, and their analysis is additionally hindered by the vast excess of other sample components. Our sample preparation methods are able to selectively separate the analytes of interest from any expected biological interferences. Finally, the highest selectivity is achieved by coupling chromatographic separation and two N-acyl chain specific selected reaction monitoring scans per analyte, enabling identification of both the N-acyl chain and the phosphatidylethanolamine moiety. The validated method is suitable for the reliable quantification of N-acylphosphatidylethanolamine species from rat brain with a lower limit of quantification of 10 pmol/g and a linear range up to 2300 pmol/g. In total, 41 N-acylphosphatidylethanolamine molecular species with six different N-acyl chains, amounting to a total concentration of 3 nmol/g, were quantified.

Determination of oxidized phosphatidylcholines by hydrophilic interaction liquid chromatography coupled to Fourier transform mass spectrometry.

A novel liquid chromatography-mass spectrometry (LC-MS) approach for analysis of oxidized phosphatidylcholines by an Orbitrap Fourier Transform mass spectrometer in positive electrospray ionization (ESI) coupled to hydrophilic interaction liquid chromatography (HILIC) was developed. This method depends on three selectivity criteria for separation and identification: retention time, exact mass at a resolution of 100,000 and collision induced dissociation (CID) fragment spectra in a linear ion trap. The process of chromatography development showed the best separation properties with a silica-based Kinetex column. This type of chromatography was able to separate all major lipid classes expected in mammalian samples, yielding increased sensitivity of oxidized phosphatidylcholines over reversed phase chromatography. Identification of molecular species was achieved by exact mass on intact molecular ions and CID tandem mass spectra containing characteristic fragments. Due to a lack of commercially available standards, method development was performed with copper induced oxidation products of palmitoyl-arachidonoyl-phosphatidylcholine, which resulted in a plethora of lipid species oxidized at the arachidonoyl moiety. Validation of the method was done with copper oxidized human low-density lipoprotein (LDL) prepared by ultracentrifugation. In these LDL samples we could identify 46 oxidized molecular phosphatidylcholine species out of 99 possible candidates.

An improved SPE method for fractionation and identification of phospholipids.

This work reports an efficient and universal SPE method developed for separation and identification of phospholipids derived from complex biological samples. For the separation step, sequential combination of silica gel-aminopropyl-silica gel SPE cartridges is applied. This setup enables separation of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylserine, cardiolipin, and sphingomyelin into four fractions according to the polarity of their headgroups. Sample acquisition of the SPE fractions is performed by a high-resolution LC-MS system consisting of a hybrid linear IT Fourier transform ion cyclotron resonance mass spectrometer coupled to RP-HPLC. The unequivocal advantage of our SPE sample preparation setup is avoidance of analyte peak overlapping in the determination step done by RP-HPLC. Overlapping phospholipid signals would otherwise exert adverse ion suppression effects. An additional benefit of this method is the elimination of polar and nonpolar (e.g. neutral lipids) contaminants from the phospholipid fractions, which highly reduces contamination of the LC-MS system. The method was validated with fermentation samples of organic waste, where 78 distinct phospholipid and sphingomyelin species belonging to six lipid classes were successfully identified.

Development and validation of an HPLC method to determine metabolites of 5-hydroxymethylfurfural (5-HMF).

The food component 5-hydroxymethylfurfural is supposed to have antioxidative properties and is therefore used as an acting agent in a novel anticancer infusion solution, named Karal®, and an oral supplementation. Previous studies showed that after oral and intravenous application, the substance is completely decomposed to its metabolites: 5-hydroxymethylfuroic acid, 2,5-furandicarboxylic acid, and N-(hydroxymethyl)furoyl glycine. The formation of a fourth metabolite, namely 5-sulphoxymethylfurfural, is still not clarified according to literature. Due to commercial unavailability, synthesis of 5-sulphoxymethylfurfural was conducted and a synthesis procedure for N-(hydroxymethyl)furoyl glycine had to be developed. Identification of the synthesised compounds was proven by LC-MS and NMR. An appropriate HPLC method was established to obtain good separation of the four possible metabolic substances and 5-hydroxymethylfurfural within 12 min via a HILIC column (150 × 4.6 mm, 5 µm) using a gradient grade system switching from mobile phase A (ACN/ammonium formate 100 mM, pH 2.35, 95:5, v/v) to mobile phase B (ACN/ammonium formate 100 mM, pH 2.35, 85:15, v/v). The procedure was afterward validated following ICH guidelines in terms of selectivity, linearity, precision, LOD, and LOQ.

A comprehensive method for lipid profiling by liquid chromatography-ion cyclotron resonance mass spectrometry.

This work aims to combine chromatographic retention, high mass resolution and accuracy, MS/MS spectra, and a package for automated identification and quantitation of lipid species in one platform for lipidomic analysis. The instrumental setup elaborated comprises reversed-phase HPLC coupled to a Fourier transform ion cyclotron resonance mass spectrometer (LTQ-FT), and Lipid Data Analyzer (LDA) software. Data analysis for lipid species quantification in this platform is based on retention time, mass resolution of 200,000, and mass accuracy below 2 ppm. In addition, automatically generated MS/MS spectra provide structural information at molecular level. This LC/MS technology allows analyzing complex biological samples in a quantitative manner as shown here paradigmatically for murine lipid droplets having a huge surplus of triacylglycerol species. Chromatographic preseparation of the bulk lipid class alleviates the problem of ion suppression of lipid species from other classes. Extension of 1D to 2D chromatography is possible, yet time consuming. The platform affords unambiguous detection of lipid species as low as 0.1‰ within major lipid classes. Taken together, a novel lipidomic LC/MS platform based on chromatographic retention, high mass resolution and accuracy, MS/MS analysis, and quantitation software enables analysis of complex samples as demonstrated for lipid droplets.

Alternative reagents for chemical noise reduction in liquid chromatography-mass spectrometry using selective ion-molecule reactions.

Reduction of ionic chemical background noise based on selective gas-phase reactions with chosen neutral reagents has been proven to be a very promising approach in liquid chromatography-mass spectrometry (LC-MS). In this study further investigations on alternative reagents including the disulfides (dimethyl disulfide, diethyl disulfide, methyl propyl disulfide), dimethyl trisulfide, ethylene oxide, and butadiene monoxide, for example, have been carried out. Tandem mass spectrometric studies of ion/molecule reactions indicate that-besides dimethyl disulfide-ethylene oxide and butadiene monoxide also exhibit very efficient reactions with background ions. Furthermore, it is confirmed that the reactions are very selective according to the test with some analyte ions. In contrast to its rapid reactions with background ions, ethylene oxide does not react, or reacts much less, with these analytes. Therefore, it can be used as an alternative reagent for noise reduction. Although reactions of the other tested neutral reagents with background ions are evaluated, they are generally not suitable as reagents for this purpose because of lack of reactivity or dramatic ion losses during reactions.

Microwave-assisted derivatization of acidic herbicides for gas chromatography-mass spectrometry.

Microwave radiation is used to speed up chemical derivatization. In the present study, three microwave-assisted techniques for the methylation of chlorophenoxy acid herbicides prior to analysis by gas chromatography coupled with mass spectrometry are compared. Derivatization was performed with the catalysts sulphuric acid and boron trifluoride as well as with trimethylsilyldiazomethane. In order to establish optimized and stable conditions, a screening for statistically significant factors by means of experimental designs was carried out and supplemented by a careful optimization. Special emphasis has been given to an accurate validation to prove the performance of the techniques. Furthermore, all microwave-assisted methods were compared with their conventional analogues. The optimized methods are valid for routine analysis of different matrices such as water, soil, sediment or tissues, especially for high sample throughput since a simultaneous derivatization of up to 64 samples in one run is possible.

Multi-residue determination of seven quinolones antibiotics in gilthead seabream using liquid chromatography-tandem mass spectrometry.

A sensitive and selective confirmatory analytical method for the multi-residue determination of seven quinolones (ciprofloxacin, enrofloxacin, sarafloxacin, danofloxacin, oxolinic acid, nalidixic acid and flumequine) in gilthead seabream (Sparus aurata) was developed. The sample pre-treatment involves extraction with 0.1 M NaOH and purification by solid-phase extraction (SPE) on Waters Oasis HLB cartridges followed by the determination of all compounds in a single LC-electrospray ionization MS/MS run. Separation was achieved on a Perfectsil ODS-2, 5 microm, 250 mm x 4 mm, analytical column (MZ Analysentechnik) by gradient elution using a mixture of 0.2% (v/v) formic acid, methanol and acetonitrile within 30 min. Multiple reaction monitoring (MRM) was used for selective detection of each quinolone. Accuracy was evaluated through recovery studies at three different fortification levels. The mean recoveries are between 90 and 132% for the selected levels with RSD values lower than 20%. The method presents satisfactory results for linearity, precision and limits of quantification. The latter are much lower than the maximum residue limits (MRLs) established by the European Union for quinolones in fish tissues (6-8 microg/kg).

Optimized derivatization of acidic herbicides with trimethylsilyldiazomethane for GC analysis.

In the present study, the preparation of methyl esters of chlorophenoxy acids with trimethylsilyldiazomethane (TMSD) is reported. TMSD is a mild and safe alternative to the highly toxic, thermally labile, and explosive diazomethane. Due to the novelty of this method, the characteristics of the reaction were investigated and the derivatization procedure was screened for statistically significant factors. The parameters identified as influential are the amount of methanol as well as reaction time and temperature. A careful optimization by a complete factorial response surface design was carried out to generate optimal reaction conditions with respect to maximum recovery and reaction yield. Special emphasis has been given to an accurate validation of the derivatization procedure. LODs were determined between 4.1 and 8.1 ng/mL for the enriched sample and linearity was proofed for more than five orders of magnitude. The applicability of the method was demonstrated by the analyses of quality control material including sample preparation with SPE. Polar herbicides investigated in the study belong to acetic acid (2,4-D, MCPA, and 2,4,5-T), butanoic acid (MCPB), and propanoic acid (mecoprop). An efficient, fast, and high-throughput method for the determination of polar herbicides including the potential for automation is proposed.

Extraction and derivatization of polar herbicides for GC-MS analyses.

A sample preparation procedure including a simultaneous microwave-assisted (MA) extraction and derivatization for the determination of chlorophenoxy acids in soil samples is presented. For a selective and sensitive measurement, an analytical technique such as GC coupled with MS needs to be adopted. For GC analyses, chlorophenoxy acids have to be converted into more volatile and thermally stable derivatives. Derivatization by means of microwave radiation offers new alternatives in terms of shorter derivatization time and reduces susceptibility for the formation of artefacts. Extraction and derivatization into methyl esters (ME) were performed with sulphuric acid and methanol. Due to the novelty of the simultaneous extraction and derivatization assisted by means of microwave radiation, a careful investigation and optimization of influential reaction parameters was necessary. It could be shown that the combination of sulphuric acid and methanol provides a fast sample preparation including an efficient clean up procedure. The data obtained by the described method are in good agreement with those published for the reference material. Finally, compared to conventional heating and also to the standard procedure of the EPA, the sample preparation time could be considerably shortened.

DDT degradation during enhanced solid-liquid extractions. A consideration.

Analysis of p,p'-DDT in solid environmental samples, like soil or sediment, requires monitoring the breakdown of this insecticide during sample preparation and measurement, which produces mainly p,p'-DDD and p,p'-DDE. The occurrence of matrix-enhanced p,p'-DDT degradation during GC injection is a well-known phenomenon; careful cleaning and pre-treatment of the GC injection port enables for an overcoming of this problem. Modern solid-liquid extraction methods apply high temperatures and/or pressures to enhance the extraction kinetics and diffusion rates of the target compounds from the solid matrix. Due to the parameters, high temperature and catalytic surface, DDT could break down during enhanced solid-liquid extraction comparable to the degradation process during GC injection. In the present study a performance evaluation by an experimental design of microwave-assisted extraction in closed vessels, pressurized-liquid extraction and fluidized-bed extraction with standard solutions of p,p'-DDT and with a quality control material was carried out. In order to identify the effect of matrix-enhanced DDT degradation during solid-liquid extraction a careful analytical protocol using isotope labelled standards was developed. At high temperatures matrix-enhanced degradation could be observed during all three investigated extraction methods. This result might have important implications on the resultant interpretations of environmental degradation of DDT.

Microwave-assisted derivatization of 2,5-hexanedione in urine: evaluation using GC-MS and GC-ECD.

2,5-Hexanedione, the main metabolite of n-hexane, can be responsible for axonal degeneration symptoms via formation of pyrrol-adducts with several amino acids. In order to make it amenable to gas chromatographic analysis, a protocol including microwave assisted derivatization is presented and compared to state-of-the-art technique of urine analysis. The applied methodology includes derivatization with O-(2,3,4,5,6-pentafluorobenzyl) hydroxylamine, extraction of the oximes and final analysis using either GC-MS or GC-muECD. Furthermore, the mass spectra of derivatized 2,5-hexanedione and 5-hydroxy-2-hexanone as well as preliminary excretion kinetics are provided. Orthogonal regression methodology demonstrated superior sensitivity for the microwave heating. Limits of detection were calculated to be approximately 20 ng mL(-1) with both MS and electron capture detection, the decompositon of excess derivatizing agent using sulfuric acid, following the reaction is beneficial. A matrix effect caused by urine was not observed, a calibration in aqueous matrix ensures accurate results therefore. Microwave heating yields excellent results regarding recovery, sensitivity and the time needed for sample preparation, furthermore, it is demonstrated that both mass selective as well as electron capture detection are of comparable suitability for this task.

Optimized extraction of polycyclic aromatic hydrocarbons from contaminated soil samples.

A comparison of Soxhlet extraction and a new extraction technique, fluidized-bed extraction, has been conducted. The extraction of polycyclic aromatic hydrocarbons (PAHs) by this new technique has been optimized considering as experimental variables the variation of the number of extraction cycles and the holding time after reaching the heating temperature by means of a surface response design. The significance of the operational parameters of the fluidized-bed extraction onto the performance characteristics has been investigated. For the determination of the analytes, a cleanup of the extracts followed by gas chromatography with mass spectrometric detection was used. The accuracy of the method was established by extraction and analysis of a reference material, supplied from the European Commission's Joint Research Centre.

Comparative study of turbulent solid-liquid extraction methods for the determination of organochlorine pesticides.

The aim of any extraction method in analytical chemistry is to effectively separate the analytes from the matrix with minimal solvent and time required. In this study, a comparison of the classical Soxhlet extraction and some new turbulent solid-liquid extraction techniques, such as fluidized-bed extraction (FBE), modified dive-in fluidized-bed extraction (dive-in FBE), modified dive-in Soxhlet extraction (dive-in SE) and dive-in thimble extraction (dive-in TE) for the determination of organochlorine pesticides (OCPs) was carried out. The turbulent extraction methods were performed by using the fexIKA vario control series extractor and by modification of the extraction system to dive-in technique, respectively. In addition, FBE and dive-in FBE were operated under the same, only for the FBE system established, optimum conditions. For the determination of the analytes a selective clean-up of the extracts followed by a gas chromatography (GC) method with mass spectrometric detection was used. All advanced extraction methods with reduced time and solvent consumption exhibited higher extraction efficiency than the standard procedure, Soxhlet extraction.

Occurrence of triazines in surface and drinking water of Liaoning Province in Eastern China.

The Liaoning Province in Eastern China has a prosperous economy and traditional agricultural background. Steadily increasing water demand implies the direct use of surface water for drinking water support and foodstuff production. Due to the extensive use, high persistence, water solubility and relatively weak adsorptivity, the chloro s-triazines contaminate the aquatic environment and are therefore regularly and most frequently detected in ground and surface water. Water sample enrichment procedures were performed by solid-phase extraction (SPE) using different materials. During a 1-year monitoring program on the Liao-He River, water sample enrichment was performed using C(18)-SPE cartridges. These SPE cartridges showed low recoveries for the atrazine metabolites, although high triazine values were detected. Based on these results, a pilot sampling on drinking water in the Liao-He region was performed. This time, in addition to the C(18)-SPE cartridges, a new polymer SPE sorbent made from a balance ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone, was used. Both materials were characterized by establishing breakthrough curves for the investigated analytes and up to large volumes.

Chemometrical classification of pumpkin seed oils using UV-Vis, NIR and FTIR spectra.

The main outcome of this work is elaboration of classification models for edible oil samples representing the most widespread brands of Austrian pumpkin seed oil. A complete spectral characterisation of the pumpkin seed oil samples by UV-Vis, NIR and FTIR spectra was obtained together with their basic sensorial classification. Chemometrical processing of the measured data enabled the detection of the most important spectral features, which are crucial for categorising the oils into two or three classes according to their sensory quality evaluated by a panel of experts. The elaborated models thus make it possible to predict the category into which a hitherto unclassified oil sample belongs--considering classification into either two categories, containing oils with overall acceptable scores or oils that were not accepted, or three categories, involving oils fulfilling all quality criteria, oils with good scores and not accepted oils. This will perspectively facilitate the determination of chemical substances responsible for bad taste, odour and colour of the respective oil brands, as well as finding substances contributing to the excellent sensorial perception of some tested products.

Determination and quantification of clonidine in human blood serum.

Clonidine ((2-[2,6-dichlorophenyl]amino)-2-imidazoline) preferentially stimulates central alpha(2)-adrenoceptors, which leads to inhibition of sympathetic tone, resulting in a lowering of arterial pressure and of heart rate. Additionally, many other desirable and undesirable effects are described, including analgesia, sedation and withdrawal reactions, which consist of a sudden rise in arterial pressure, nervousness, agitation and increased heart rate. The present study has the goal to develop a simple and effective method for the analysis of trace amounts of clonidine in human blood serum. Special emphasis is necessary to make application of electron impact ionization and separation of the analyte fragments in a quadruple mass analyzer suitable. The procedure comprises solid phase extraction followed by formation of the pentafluorobenzyl derivative. Further purification is achieved by phase transfer extraction into an acidic aqueous solution succeeded by re-extraction into dichloromethane. After solvent exchange, an aliquot is injected into the gas chromatograph equipped with a DB5 MS capillary column and a mass spectrometric detector. Chromatograms are recorded in single ion monitoring mode. Quantification is accomplished by internal standardization with moxonidine [4-chloro-5-(2-imidazolin-2-yl-amino)-6-methoxy-2-methylpyridine].

Triazines in the aquatic systems of the Eastern Chinese Rivers Liao-He and Yangtse.

The results of a one-year monitoring program on the two Eastern Chinese River systems, i.e. the Liao-He and the Yangtse, with special emphasis on the presence of triazine herbicides are presented. Sediment, suspended solids and water samples from both rivers were analyzed. Additionally, recovery experiments on the SPE-in-field-enrichment procedure and the extraction methods were performed. The samples were measured by gas chromatography coupled with mass spectrometry, electron capture detection and a newly developed mu-plasma atomic emission detector. A typical result of a one-year monitoring was obtained in case of the Liao-He: During winter, at low water period, low triazine values were found. A similar situation was found in early spring. Highest concentrations of atrazine up to 1600 ng/l were found in late spring in the water samples. Maximum concentrations of atrazine, simazine, propazine, simetryn and prometryn were observed in this season as a result of the actual use of triazines. Finally, after the high water period in autumn the triazine concentrations decreased. Additionally, atrazine adsorbed on sediment (up to 2.8 ng/g) and suspended solids was determined (up to 8 ng/l) during late spring sampling. Therefore, the logarithm of the organic carbon based sorption coefficient of atrazine could be calculated. Low levels of atrazine were measured in the water of Yangtse (up to 18.3 ng/l). The concentrations from all sampling points and sampling stations of a particular sampling date were similar, which indicates a homogeneous distribution of this herbicide. Due to the high discharge rate of up to 79,000 m3/s in case of the Yangtse a considerable mass transport of up to 57.5 kg per day atrazine may take place, even at concentrations below the European drinking water limit of 100 ng/l.

Study of the influence of maceration time and oenological practices on the aroma profile of Vranec wines.

Vranec is one of the most important red grape varieties in Republic of Macedonia, grown in all vineyards, mostly in the Tikves wine region. In this study, Vranec wines produced with different maceration times (4, 7, 14 and 30 days) in presence of enzyme and oak chips during fermentation were studied in order to determine the influence of vinification conditions on the aroma profile. The volatile compounds were determined using headspace solid phase microextraction (HS-SPME) with a PDMS/Carboxen/DVB fibre, coupled with gas chromatography-mass spectrometry (GC-MS). In total 63 aroma compounds were detected revealing a complex aroma profile of Vranec wines composed of esters, alcohols, fatty acids, aldehydes, ketones and sulphur compounds. The content of aroma compounds was related mostly to maceration time, observing increased relative amount of alcohols, esters and fatty acids from the fourth to seventh day of maceration and the presence of oak chips during the fermentation enhanced their formation. The Student-Newman-Keuls test has been applied to ascertain possible significant differences between the studied wines, and principal component analysis has been employed, showing separation and grouping of the wines according to maceration time and oak chips treatment.

Optimization and application of microwave-assisted acid hydrolysis for rapid quantification of protein oxidation markers using LC-MS.

Simple and efficient microwave-assisted acid hydrolysis (MAAH) of proteins was used for rapid quantification of α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) as major protein oxidation markers. The precursor amino acid residues corresponding to AAS and GGS in oxidized proteins were derivatized by reductive amination with sodium cyanoborohydride (NaCNBH(3)) and p-aminobenzoic acid (ABA) followed by MAAH to generate the marker derivatives AAS-ABA and GGS-ABA. The quantification was performed using electrospray ionization liquid chromatography-mass spectrometry (ESI LC-MS). The important parameters for hydrolysis were optimized, which include the temperature, the reaction time, the acid concentration and volume as well as the microwave power. Compared to the conventional acid hydrolysis of 18-24h using 6-12 M HCl at 110°C applied commonly in the literature and also in this work, MAAH of proteins can be completed as fast as in only 2-10 min and, additionally, with a 3-5 times higher yield of the final derivatization products. Furthermore, a better agreement between the ratio of the detected derivatization products and the theoretical yields from the studied protein has also been achieved, which indicates that MAAH may serve as a more reliable method of acid hydrolysis for this purpose than that with conventional thermal heating. The MAAH method is demonstrated to be a time-saving, reproducible and efficient technique for studying AAS and GGS as protein oxidation markers using LC-MS.