Gas chromatographic-mass spectrometric determination of alkylphosphonic acids from aqueous samples by ion-pair solid-phase extraction on activated charcoal and methylation

In the present paper, we report an improved ion-pair solid-phase extraction (IP-SPE) method for the analysis of alkylphosphonic acids, namely, methyl, ethyl and propylphosphonic acids, present in the aqueous sample. The aqueous sample was mixed with an ion-pair reagent, phenyltrimethylammonium hydroxide (PTMAH) and passed through activated charcoal SPE cartridge. The retained chemicals in the cartridge were extracted with methanol and analysed by gas chromatography-mass spectrometry (GC-MS) under the electron impact ionization (EI) mode. The analytes were converted to their methyl esters by pyrolytic methylation in the hot GC injection port. The recoveries of alkylphosphonic acids were above 95% and the minimum detection limits were as low as 10 ng/mL. The recovery of the test chemicals was tested with solvents, dichloromethane, n-hexane, ethyl acetate, acetone, acetonitrile and methanol. The chemicals could be efficiently extracted by the hydrophilic solvents. The method did not work at the highly acidic pH (when acidified with dilute HCl) but worked well from pH 4.0 to 14.0. The present method was also tested with other tetra-(methyl, ethyl, propyl and n-butyl)ammonium hydroxides. The test chemicals were not converted to their methyl and ethyl esters with tetramethyl and tetraethylammonium hydroxides, whereas they were converted to their corresponding propyl and n-butyl esters with tetrapropyl and tetra(n-butyl)ammonium hydroxides. The method was also applied to two highly cross-linked polymeric sorbents DSC-6S and Oasis HLB. The recovery of the chemicals on these sorbents was observed to be poor. Methylation using phenyltrimethylammonium hydroxide is non-hazardous and advantageous over methylation using diazomethane. The method was applied to the analysis of aqueous samples given in one of the official proficiency tests conducted by the Organization for the Prohibition of Chemical Weapons and all the spiked chemicals were identified as methyl esters.     

Reaction efficiency of organic alkalis with various classes of lipids during thermally assisted hydrolysis and methylation

Reaction efficiencies of two organic alkalis, tetramethylammonium hydroxide (TMAH) and trimethylsulfonium hydroxide (TMSH), with lipids during thermally assisted hydrolysis and methylation (THM) were examined focusing on (1) the types of lipids and (2) degree of unsaturation of fatty acid moieties. Different types of lipids such as triglycerides, phospholipids, free fatty acids and cholesteryl esters containing saturated, monounsaturated or polyunsaturated fatty acid (PUFA) residues were subjected to THM-gas chromatography (GC) in the presence of TMAH or TMSH. The obtained results revealed that the THM reaction using TMAH allowed almost quantitative methylation of saturated and monounsaturated fatty acid components independently of the classes of lipids. However, strong alkalinity of TMAH brought about isomerization and/or degradation of PUFA components. In contrast, the use of TMSH was effective to highly sensitive detection of PUFA as well as saturated and monounsaturated fatty acid components contained in triglycerides, phospholipids (phosphatidylcholines) and free fatty acids. On the other hand, TMSH was proved to react hardly with any kind of fatty acid residues in cholesteryl esters due to their steric hindrance.     

Separation and identification of isomeric acidic degradation products of organophosphorus chemical warfare agents by capillary electrophoresis-ion trap mass spectrometry

Capillary electrophoresis (CE) coupled to ion trap mass spectrometry (MS) was evaluated for the separation and identification of chemical warfare agent degradation products (alkylphosphonic acids and alkyl alkylphosphonic acids). Different analytical parameters were optimized in negative ionization mode such as electrolyte composition (15 mM CH(3)COONH(4), pH 8.8), sheath liquid composition (MeOH/H(2)O/NH(3), 75:25:2, v/v/v), nebulization and ion trapping conditions. A standard mixture of five alkylphosphonic (di)acids and five alkyl alkylphosphonic (mono)acids containing isomeric compounds was used in order to evaluate CE selectivity and MS identification capability. The obtained electropherograms revealed that CE selectivity was very limited in the case of alkyl alkylphosphonic acid positional isomers, whereas isomeric isopropylphosphonic and propylphosphonic acids were baseline-separated. CE-MS-MS experiments provided an unambiguous identification of each isomeric co-migrating alkyl alkylphosphonic acids thanks to the presence of specific fragment ions. On the other hand, CE separation was mandatory for the identification of isomeric alkylphosphonic acids, which led to the same fragment ion and could not be differentiated by MS-MS. The developed method was applied to the analysis of soil extracts spiked with the analytes (before or after extraction treatment) and appeared to be very promising since resolution and sensitivity were similar to those observed in deionized water. Especially, analytes were detected and identified in soil extract spiked at 5 microg mL(-1) with each compound before extraction treatment.     

Quantification of organic acids in particulate matter by coupling of thermally assisted hydrolysis and methylation with thermodesorption-gas chromatography–mass spectrometry

A quantitative method for the determination of organic acids in atmospheric particles is developed. The method couples a derivatisation step (thermally assisted hydrolysis and methylation) and a Curie point pyrolyser as a thermal desorption technique and gas chromatography–mass spectrometry (CPP-GC–MS). Among the reagents tested (tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAAc) and phenyltrimethylammonium hydroxide (TMPAH)), the best performance was found using TMAAc as a derivatisation reagent for the reaction time of 4 s at 510 °C as heating temperature. Calibration was performed for a series of fatty acids (FA), dicarboxylic acids (DCA) and terpenoic acids (TA) under these conditions. Coefficients of determination (R²) were between 0.94 and 0.98. Limits of detection (LOD) were in the nanogram-range between 0.1 and 3.6 ng. The method is applied on atmospheric particle samples to obtain the quantification reproducibility and quantification limits. Reproducibility was determined in terms of relative standard deviations (RSD) for ambient aerosol samples collected by a high-volume-sampler (HVS, RSD = 6–45%, n = 10) and a Berner impactor (BI, RSD = 5–34%, n = 10). Based on 24 h sampling time the developed method enables quantification of all three classes of acids for both sampling techniques. Calibration data and presented volume concentrations are compared with literature data. A comparison with an off-line methylation-GC–MS using BF₃ as a derivatisation reagent and capillary electrophoresis coupled mass spectrometry (CE-MS) showed a good agreement. Minimal sample preparation is the main advantage of the developed method. Depending on the sensitivity requirements the present method can be a fast and simple alternative to GC–MS techniques with conventional sample preparation steps for semi-volatile organic acids.     

Determination of the carbon isotopic composition of whole/intact biological specimens using at-line direct thermal desorption to effect thermally assisted hydrolysis/methylation

In this paper, we discuss the use of a direct thermal desorption (DTD) interface as an alternative to Curie-point flash pyrolysis system as an inlet technique in gas chromatography-combustion isotope-ratio mass spectrometry (GC/C-IRMS) analysis of whole/intact phytoplankton and zooplankton specimens. The DTD in combination with a combipal auto-injector is programmed to perform the injection, evaporation of solvents, transport of capped programmed-temperature vaporizer (PTV) liners to the PTV injector and chemical derivatisation (thermally assisted hydrolysis/methylation; THM) such that a profile of a cellular fatty acids is obtained. Flow-cytometric sorted microalgae and handpicked zooplankton are used as samples with trimethylsulfonium hydroxide (TMSH) as methylating reagent. A major advantage of this novel approach over the Curie-point technique is the automation of the total procedure, which allows unattended analysis of large sample series. The profiles and delta(13)C carbon isotopic signatures of the fatty acid methyl esters (FAMEs) produced are very similar to those obtained using the Curie-point flash pyrolysis method. It is shown that algal samples must be kept no longer than 48 h in the DTD sample tray prior to the THM-analysis in order to maintain the integrity of their FAME profile.     

Derivatisation in gas chromatographic determination of acidic herbicides in aqueous environmental samples

This paper reviews derivatisation processes applied in chromatographic determination of acidic herbicides (with carboxyl and phenol groups), mainly in aqueous environmental samples. The discussion focuses on the basic derivatisation reactions used to convert herbicides to derivatives to make them analysable by gas chromatography, and possibly to reduce detection limits and/or increase extraction recovery from aqueous samples. The reactions are transesterification, esterification, silylation, alkylation, and extractive and pyrolytic alkylation. The reagents used to conduct the reactions are numerous. Diazomethane is a very efficient methylation reagent but explosive and toxic. Methyl iodide also ensures rapid and efficient methylation. Benzyl bromide can be used directly in water but derivatisation yield is low and reproducibility is poor. Butyl chloroformate and dimethyl sulfite can also be used for derivatisation in water, and acetic anhydride can be used for in-situ derivatisation of phenolic herbicides. For increasing selectivity of GC detection pentafluorobenzyl bromide (for ECD) and (2-cyanethyl)dimethyl(diethylamino)silane (for NPD) have been applied. Very characteristic ions are produced in mass spectra if silyl groups are introduced, e.g. by using bis(trimethylsilyl)trifluoroacetamide. Tetramethylammonium, trimethylphenylammonium, tetraalkylammonium, and trimethylsulfonium hydroxides and salts can by used for derivatisation at elevated temperature in the GC injection port. Extractive alkylation is relatively efficient if tetraalkylammonium salts with long chain alkyl groups are used.     

树脂酸性组分的氢氧化四甲铵甲基化与GC-MS分析

利用氢氧化四甲铵(TMAH)对马尾松树脂(塔罗油)进行甲基化，并对其产物进行GC—MS分析；研究表明TMAH甲基化后进行GC—MS分析是测定树脂中酸性组分的一种快速有效的方法，但酸性组分中的脂肪酸要比树脂酸难于甲基化，因此为了确保甲基化反应完全，应适当延长甲基化反应时间。     

Fast identification of mycobacterium species by GC analysis with trimethylsulfonium hydroxide (TMSH) for transesterification

Trimethylsulfonium hydroxide (TMSH) reproducibly converts fatty acids bound in, e.g., biomolecules such as phospholipids and/or glycerides, into the corresponding fatty acid methyl esters (FAMEs). The transesterification can be performed at room temperature in a fast single step reaction. Surprisingly, secondary alcohols and mycolic acid cleavage products (MACPs) are also released from mycobacteria under these conditions. The complex reaction mixtures containing FAMEs, MACPs, and secondary alcohols can easily be separated by high resolution temperature-programmed capillary GC. Different species of mycobacteria give rise to characteristic chromatographic patterns and the amount of lipids from a single colony of mycobacteria is sufficient for reliable identification of the bacteria. The profiles of the chromatograms match well those obtained from other sample preparation techniques. The TMSH method of identification of mycobacteria from the patterns of the gas chromatograms is faster and more sensitive than conventional methods, which also involve transesterification. The identification of mycobacterial species by microbiological culture techniques is difficult to perform and requires several weeks.     

Identification of non-cross-linked compounds in methanolic extracts of cured and aged linseed oil-based paint films using gas chromatography-mass spectrometry

Methanolic extracts of paint samples of different composition and age were qualitatively investigated by GC–MS using an on-column injector after off-line methylation or trimethylsilyl derivatisation, and on-line thermally assisted (trans)methylation with tetramethylammonium hydroxide using Curie-point pyrolysis–GC–MS. The combination of these three analytical strategies led to the identification of typical oxidation products of unsaturated fatty acids by interpretation of their mass spectrum. Some of the identified compounds have not been reported before. Both the off-line and on-line GC–MS strategy show series of short-chain fatty (di)acids and C₁₆ and (oxidised) C₁₈ fatty acids. The major advantage of the on-line pyrolysis–GC–MS approach is that chemical work-up is minimal and very quick. With this technique both the carboxylic acid functionalities, and hydroxyl groups are methylated. Young paint films are shown to contain relatively more oxidised C₁₈ fatty acids and less diacids compared to older paints, which is indicative for the on-going oxidation processes within the paint. After trimethylsilylation, monoacylglycerols are detected indicative for hydrolytic processes, which reflect the relative distribution of the most prominent silylated fatty acids present. Relatively more C₁₆ and C₁₈ monoacylglycerols are found in young paints, whereas older paints contain higher amounts of monoacylglycerols of diacids.     

Trans-esterification of fatty acids from microorganisms and human blood serum by trimethylsulfonium hydroxide (TMSH) for GC analysis

Summary Trimethylsulfonium hydroxide (TMSH) can convert fatty acids into the corresponding fatty acid methyl esters (FAMEs) in a single step. These fatty acids may also be bound in biomolecules such as phospholipids and/or glycerides. Complex mixtures of saturated and unsaturated FAMEs which may contain hydroxy and cylopropyl groups are obtained by trans-esterification; they can easily be separated in most cases by capillary GC. When FAMEs are generated from different microorganisms e.g. bacteria the patterns of the chromatograms are characteristic. Examples of characteristic patterns of bacteria with different cell wall structures are shown. The described method of transesterification can also be applied directly to blood serum without sophisticated sample pretreatment. The profiles of the chromatograms match well those described in the literature obtained by other methods of trans-esterification or sample preparation.     

Field-amplified sample stacking for the detection of chemical warfare agent degradation products in low-conductivity matrices by capillary electrophoresis-mass spectrometry

Preconcentration of chemical warfare agent degradation products (alkylphosphonic acids and alkyl alkylphosphonic acids) in low-conductivity matrices (purified water, tap water and local river water) by field-amplified sample stacking (FASS) was developed for capillary electrophoresis (CE) coupled to ion trap mass spectrometry. FASS was performed by adding a mixture of HCOONH(4) and NH(4)OH in appropriate concentrations to the sample. This allowed to control the conductivity and the pH of the sample in order to obtain FASS performances that are independent of analyte concentration. The influence of different parameters on FASS (sample to background electrolyte (BGE) conductivity ratio, injection volume and concentration of BGE) was studied to determine the optimal conditions and was rationalized by using the theoretical model developed by Burgi and Chien. A good correlation was obtained between the bulk electroosmotic velocity predicted by this model and the experimental value deduced from the migration time of the electroosmotic flow marker detected by mass spectrometry (MS). This newly developed method was successfully applied to the analysis of tap water and local river water fortified with the analytes and provided a 10-fold sensitivity enhancement in comparison to the signal obtained without preconcentration procedure. The quite satisfactory repeatability and linearity for peak areas obtained in the 0.5-5 microg mL(-1) concentration range allow quantitative analysis to be implemented. Limits of detection of 0.25-0.5 microg mL(-1) for the alkyl alkylphosphonic acids and of 0.35-5 microg mL(-1) for the alkylphosphonic acids were reached in tap water and river water.     

Direct chemolysis-gas chromatography-mass spectrometry for analysis of paint materials

A novel direct method using (m-trifluoromethylphenyl)trimethylammonium hydroxide (TFTMAH) has been developed for on-line (trans)esterification of lipidic materials (drying oils, egg) prior to GC-MS analysis. The method was first optimised by comparing three N-methylammonium hydroxide reagents (TMAH, PhTMAH, TFTMAH) and triolein as a lipid standard. Secondly, the procedure was tested on a series of fresh and naturally aged (up to 40 years) (un)pigmented drying oils and egg yolk reference film standards. Thirdly, the method was applied to the analysis of a sample from an altar painting "Resurrection of Christ" by Francesco Solimena (1723) from the Chapel of Upper Belvedere in Vienna. The relative proportions of the fatty acids from the translucent size layer confirm the presence of partially degraded oil, probably prepolymerised walnut oil.     

Solid-phase microextraction and on-line methylation gas chromatography for aliphatic carboxylic acids

Methylation of carboxylic acids upon syringe injection of a mixture of the acid sample and phenyltrimethylammonium hydroxide (PTMAH) into the GC injection port is a convenient but under-utilized derivatization procedure. To minimize potential instrumental problems due to the sample matrix, it was shown that solid-phase microextraction (SPME) is effective for the absorption of both the carboxylic acid (RCOOH) and PTMAH permitting on-line methylation from the fiber. A comparison of three fibers, polydimethylsiloxane (PDMS), polyacrylate (PA), and carboxene/PDMS for decanoic and stearic acids showed the carboxene/PDMS fiber was about five times more effective for the extraction of the RCOOH-PTMAH mixture dissolved in methanol. The optimum fiber absorption time was about 20 min and the optimum desorption time in the injection port held at 280 degrees C was about 5-10 min. The optimum PTMAH/RCOOH ratio was about 125:1. Linearity for C18:0 at 3.3 x 10(-6)-3.3 x 10(-4) M was demonstrated by GC-MS with a detection limit of 1 microM. This SPME method is also effective for the methylation of C18:1, C18:2, and C18:3 fatty acids. Transesterification of olive oil using PTMAH and then on-line methylation either by the syringe method or by SPME gave comparable fatty acid methyl ester profiles.     

Evaluation of automated sample preparation, retention time locked gas chromatography-mass spectrometry and data analysis methods for the metabolomic study of Arabidopsis species

In this paper, automated sample preparation, retention time locked gas chromatography-mass spectrometry (GC-MS) and data analysis methods for the metabolomics study were evaluated. A miniaturized and automated derivatisation method using sequential oximation and silylation was applied to a polar extract of 4 types (2 types×2 ages) of Arabidopsis thaliana, a popular model organism often used in plant sciences and genetics. Automation of the derivatisation process offers excellent repeatability, and the time between sample preparation and analysis was short and constant, reducing artifact formation. Retention time locked (RTL) gas chromatography-mass spectrometry was used, resulting in reproducible retention times and GC-MS profiles. Two approaches were used for data analysis. XCMS followed by principal component analysis (approach 1) and AMDIS deconvolution combined with a commercially available program (Mass Profiler Professional) followed by principal component analysis (approach 2) were compared. Several features that were up- or down-regulated in the different types were detected.     

Identification of Indigo Dyes in Painting Layers by Pyrolysis Methylation and Silylation. A Case Study: “The Dinner of Emmaus” by G. Preti

Reactive pyrolysis, under methylating or silylating conditions, in combination with gas chromatography-mass spectrometry (GC-MS) was evaluated as an analytical method for the detection of indigo dyes in painting layers. Samples with the addition of tetramethylammonium hydroxide (TMAH) or hexamethyldisilazane (HMDS), for pyrolysis/methylation and pyrolysis/silylation experiments, respectively, were pyrolysed at 600 °C by means of a heated filament pyrolyser and the evolved products were analysed on-line by GC-MS. Methyl and silylated pyrolytic markers, related to 2-aminobenzoic acid and the indoxyl moiety, were established from the analysis of synthetic indigo, neat or as a pigment of artificial painting layers containing a siccative oil. The occurrence of these markers was investigated in a real sample, taken from the painting The dinner of Emmaus by G.Preti (XVII century), in order to identify the blu pigment. Positive identification of indigo was achieved by pyrolysis/silylation and confirmed by RAMAN spectroscopy.     

Automated Trimethyl Sulfonium Hydroxide Derivatization Method for High-Throughput Fatty Acid Profiling by Gas Chromatography–Mass Spectrometry

Fatty acid profiling on gas chromatography–mass spectrometry (GC–MS) platforms is typically performed offline by manually derivatizing and analyzing small batches of samples. A GC–MS system with a fully integrated robotic autosampler can significantly improve sample handling, standardize data collection, and reduce the total hands-on time required for sample analysis. In this study, we report an optimized high-throughput GC–MS-based methodology that utilizes trimethyl sulfonium hydroxide (TMSH) as a derivatization reagent to convert fatty acids into fatty acid methyl esters. An automated online derivatization method was developed, in which the robotic autosampler derivatizes each sample individually and injects it into the GC–MS system in a high-throughput manner. This study investigated the robustness of automated TMSH derivatization by comparing fatty acid standards and lipid extracts, derivatized manually in batches and online automatically from four biological matrices. Automated derivatization improved reproducibility in 19 of 33 fatty acid standards, with nearly half of the 33 confirmed fatty acids in biological samples demonstrating improved reproducibility when compared to manually derivatized samples. In summary, we show that the online TMSH-based derivatization methodology is ideal for high-throughput fatty acid analysis, allowing rapid and efficient fatty acid profiling, with reduced sample handling, faster data acquisition, and, ultimately, improved data reproducibility.     

In situ derivatisation using pressurized liquid extraction to determine phenols, sterols and carboxylic acids in environmental samples and microbial biomasses

Pressurized liquid extraction was combined with in-situ derivatisation to extract polar analytes such as phenols (including chlorophenols) sterols and carboxylic acids from environmental and microbial samples. This one-step protocol uses acetic anhydride as an acetylation agent, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) as an silylation agent, and boron trifluoride-methanol, phenyltrimethyl ammoniumhydroxide and trimethyl sulfoniumhydroxide as methylation agents. It results in faster extraction rates and better or comparable extraction efficiencies when compared to classical approaches. The addition of a silylation agent also facilitates the extraction kinetics of analytes not accessible to silylation (e.g. polycyclic aromatic hydrocarbons or alkylbenzenes). This may be attributed to a dissociative action of the agent to weaken analyte-matrix interactions.     

Advances in identification of plant gums in cultural heritage by thermally assisted hydrolysis and methylation

Plant gums are present in works of art as binding media for watercolours and adhesives for cellulosic substrates. Thermally assisted hydrolysis and methylation (THM) in combination with analytical pyrolysis coupled to GC/MS has been applied to the characterisation of plant gums typically used in artworks. THM products from standard samples of arabic gum, tragacanth gum and cherry gum were characterised. The main products identified are permethylated and partially methylated aldonic acids, characteristic of specific epimeric sugars. Aldonic acids were formed by alkaline hydrolysis of free reducing sugars and of reducing polysaccharide terminal groups, while methylation occurs during pyrolysis. The presence of these characteristic markers allows gum identification. A systematic analysis of all the parameters that can affect the marker yields was performed. In particular, the influence of pyrolysis temperature, reagent concentration and contact time between tetramethylammonium hydroxide and sample were studied, and different kinds of sample preparation procedures were tested. Some analyses on real watercolours were performed, and gum binders were classified using the peak area ratio of the main monosaccharide markers.     

Use of an injection port for thermochemolysis–gas chromatography/mass spectrometry: Rapid profiling of biomaterials

A simple and direct approach was developed for thermochemolytic analysis of a wide range of biomolecules present in plant materials using an injection port of a gas chromatograph/mass spectrometer (GC/MS) and a novel solids injector consisting of a coiled stainless steel wire placed inside a modified needle syringe. Optimum thermochemolysis (or Thermally Assisted Hydrolysis/Methylation) was achieved by using a suitable methanolic solution of trimethylsulfonium hydroxide (TMSH) or tetramethylammonium hydroxide (TMAH) with an injection port temperature of 350 °C. Intact, methylated flavonoids, saccharides, phenolic and fatty acids, lignin dimers and diterpene resin acids were identified. Samples include tea leaves, hemicelluloses, lignin isolates and herbal medicines. Unexpected chromatographic results using TMAH reagent revealed the presence of intact methylated trisaccharides (658 Da) and structurally informative dimer lignin markers.     

Characterisation of Siccative Oils, Resins and Pigments in Art Works by Thermochemolysis Coupled to Thermal Desorption and Pyrolysis GC and GC-MS

Thermal desorption in the presence of tetramethylammonium hydroxide (TMAH) has been applied to materials belonging to the artistic field to verify the effectiveness of the derivatisation reaction. Thermal desorption of siccative oils and abietane resins give consistent results with traditional pyrolysis in methylation conditions, while as regard antraquinonic dyes the milder thermal treatment leads to the formation of more diagnostic compounds than pyrolysis.