Pyrolysis-GC/MS and TG/MS study of mediated laccase biodelignification of Eucalyptus globulus kraft pulp

Biobleaching studies using laccase mediator system (LMS) were carried out, under optimized conditions, on two unbleached Eucalyptus globulus kraft pulps, one produced by conventional way, with kappa number of 16.1, and another with kappa number of 14.5, obtained by modified kraft procedure with a high liquor/wood ratio and with black liquor replacement in the middle of the cooking. The pulp properties before and after LMS and alkaline extraction were evaluated in terms of kappa number, hexeneuronic acid content, viscosity, brightness and acid insoluble lignin content.The original milled wood sample and the kraft pulps were characterized by pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and thermogravimetry/mass spectrometry (TG/MS). Eucalypt wood lignin produces guaiacol and syringol derivatives during pyrolysis. These lignin products can be detected with high sensitivity using the selected ion chromatograms even in the bleached pulp of low lignin content (about 0.5%). Py-GC/MS revealed that the lignin moieties were similarly altered during biobleaching as during pulping, which is exemplified by the preferential removal of aldehyde groups from the alkyl side groups. Semi-quantitative analysis of the pyrograms indicates that the lignin content of the biobleached pulps is reduced by about half in comparison with the unbleached pulps. The TG/MS results show that the hemicellulose content of wood was strongly modified during pulping resulting in higher thermal stability.     

Analysis of pitch deposits produced in kraft pulp mills using a totally chlorine free bleaching sequence

Two organic deposits accumulated in a Kraft pulp mill during pulping of Eucalyptus globulus wood and throughout a TCF (totally chlorine free) bleaching sequence were characterized. One deposit was collected after cooking and an oxygen delignification stage while the other was collected after bleaching with hydrogen peroxide. The deposits were Soxhlet extracted with acetone, and the extracts redissolved in chloroform and subsequently analyzed by gas chromatography (GC) and GC-mass spectrometry (MS) using short and medium length high temperature capillary columns, respectively. On the other hand, the insoluble residues left after the acetone extraction were analyzed by Curie-point flash pyrolysis-GC-MS and by pyrolysis-methylation-GC-MS. The compounds identified in the deposits arise from the E. globulus wood lipophilic extractives that survive the pulping and bleaching processes. Triglycerides were completely hydrolyzed during the Kraft cooking and the fatty acids dissolved. Steroids (alcohols, hydrocarbons, ketones and esters) and waxes were the main components in the deposit collected after the oxygen delignification stage. After the bleaching with hydrogen peroxide, content of the waxes were reduced and fatty acids appeared. High amounts of fatty acids salts were also identified in the deposit collected after the oxygen stage, and in minor amounts in the deposit collected after hydrogen peroxide bleaching. In contrast, this deposit was mainly made up of high amounts of lignin-derived phenolic moieties.     

Identification of intact long-chain p-hydroxycinnamate esters in leaf fibers of abaca (Musa textilis) using gas chromatography/mass spectrometry

The study of acetone-extractable components from the leaf fibers of the non-wood plant abaca (Musa textilis) resulted in the isolation and identification of series of intact hydroxycinnamate esters consisting of ferulic and p-coumaric acids esterified to long-chain fatty alcohols (C20 to C28) and omega-hydroxyfatty acids (C22 to C28). These series of compounds were characterized by high-temperature gas chromatography/mass spectrometry (GC/MS) using capillary columns (12 m length) with thin films that allowed the analysis of intact (i.e., without prior saponification) hydroxycinnamate esters. Characterization of intact individual compounds was achieved based on the mass spectra obtained by GC/MS of the underivatized compounds and their methyl and/or trimethylsilyl ether derivatives.     

Cellulose Depolymerisation and Paper Properties in E. Globulus Kraft Pulps

The aim of this work was to study the impact of cellulose depolymerisation on the beating potential and handsheet properties of the portuguese E. globulus kraft pulp. A homogeneous sample of eucalypt wood chips was cooked using different kraft pulping conditions (cooking temperatures and times, and sodium hydroxide and sodium sulphide concentrations) in order to obtain a wide variation for intrinsic viscosity of the pulps. In the range of industrial cooking conditions, this property was found to be linearly dependent on the effective alkali charge, for a given cooking time and temperature. Unbeaten and beaten (at 2000 rev. PFI) pulp properties were evaluated and the results confirm that the higher the pulp intrinsic viscosity the better the pulp beatability and the paper properties. However, the differences in the latter cannot be exclusively explained by the differences in viscosity, since pulps with the same viscosity may exhibit distinct papermaking potentials. It was then necessary to scan other pulp chemical characteristics that could also influence the development of paper properties such as lignin, pentosan content and polysaccharides relative composition.     

Comprehensive two-dimensional gas chromatography, retention indices and time-of-flight mass spectra of flavonoids and chalcones

The applicability of comprehensive two-dimensional gas chromatography (GC×GC) for flavonoids analysis was investigated by separation and identification of flavonoids in standards, and a complex matrix natural sample. The modulation temperature was optimized to achieve the best separation and signal enhancement. The separation pattern of trimethylsilyl (TMS) derivatives of flavonoids was compared on two complementary column sets. Whilst the BPX5/BPX50 (NP/P) column set offers better overall separation, BPX50/BPX5 (P/NP) provides better peak shape and sensitivity. Comparison of the identification power of GC×GC-TOFMS against both the NIST05 MS library and a laboratory (created in-house) TOFMS library was carried out on a flavonoid mixture. The basic retention index information on high-performance capillary columns with a non-polar stationary phase was established and database of mass spectra of trimethylsilyl derivatives of flavonoids was compiled. TOFMS coupled to GC×GC enabled satisfactory identification of flavonoids in complex matrix samples at their LOD over a range of 0.5-10 μg/mL. Detection of all compounds was based on full-scan mass spectra and for each compound a characteristic ion was chosen for further quantification. This study shows that GC×GC-TOFMS yields high specificity for flavonoids derived from real natural samples, dark chocolate, propolis, and chrysanthemum.     

Detection and identification of quinonemethide triterpenes in Peritassa campestris by mass spectrometry

Analysis of tingenone and tingenol quinonemethide triterpenes was made by gas chromatography/mass spectrometry (GC/MS) of their trimethylsilyl (TMS) ethers. An extra TMS group, in addition to those predicted from the known structures, is added to these compounds during the derivatization process. The electron impact mass spectra showed base peaks at m/z 549 and 623, respectively, for the TMS derivatives of tingenone and tingenol, and electrospray (ES) and collision-activated dissociation (CAD) studies indicate that these ions correspond to losses of a methyl group from the derivatives studied. A mechanism, based on ES-MS/MS studies, is suggested for the derivatization and fragmentation pattern.     

Analysis of impurities occurring in a totally chlorine free-bleached Kraft pulp

A set of impurities (specks) occurring in a TCF (totally chlorine free)-bleached Kraft pulp of Eucalyptus globulus wood was studied. The impurities were Soxhlet extracted with acetone, and the extracts subsequently analyzed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS) using high-temperature capillary columns. The lipophilic fraction isolated from E. globulus wood extractives was also analyzed for comparison. The composition of the acetone extracts was very similar to that of E. globulus wood. Fatty acids, steroid hydrocarbons, sterols, steroid ketones and sterol esters, arising from E. globulus wood extractives survived the cooking and bleaching processes and accumulated in the pulp. On the other hand, the residue left after acetone extraction was studied by pyrolysis–GC–MS. The results indicated that it was composed of small particles of polyisoprene rubber. In conclusion, the speck impurities studied here seems to be composed of two different moieties, a lipophilic part arising from wood extractives and a core of small particles of synthetic polymers (polyisoprene rubber).     

Oxime-trimethylsilylation method for analysis of wood pyrolysate

Oxime-trimethylsilylation (TMS) method was applied to the analysis of wood pyrolysate. Quantitative determination of hydroxycarbonyls such as glycolaldehyde, which are important pyrolysis products of wood polysaccharide, is difficult as indicated by the gas chromatography–mass spectrometry (GC–MS) and ¹H NMR analysis of glycolaldehyde. Glycolaldehyde decomposed into formic acid and the unknown compound with molecular weight of 72 at the injector in its GC analysis. ¹H NMR analysis of glycolaldehyde in acetone-d₆ indicated the complex mixture with its dimerization products. Glycolaldehyde was quantified precisely as oxime-TMS derivative (E/Z-mixture) of the monomer by GC after oximation with hydroxylamine hydrochloride and the following trimethylsilylation with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). With this method, the other carbonyls such as furfural, 5-hydroxymethylfurfural and hydroxyacetone could also be determined as their oxime-trimethylsilylated derivatives. Furthermore, anhydrosugars such as levoglucosan and levomannosan in wood pyrolysate were also determined, simultaneously, as their TMS derivatives. Finally, oxime-TMS method is proposed as a quantification method of the pyrolysis products derived from wood polysaccharide.     

Analytical pyrolysis study of biodelignification of cloned Eucalyptus globulus (EG) clone and Pinus pinaster Aiton kraft pulp and residual lignins

This study centred on the analysis of lignin in situ of cloned eucalypt and pine kraft pulps. Trametes versicolor laccase-violuric acid system (LMS) delignifications were performed on a softwood (Pinus pinaster) and a hardwood (Eucalyptus globulus) conventional kraft pulp with an initial kappa number of 34.5 and 15.5, respectively. The LMS treated pulps were then subjected to alkaline extraction stages (E). The kappa number data show that LMS is effective at biodelignifying both softwood and hardwood kraft pulps. However, under the conditions employed in this study, a greater level of biodelignification was obtained with LMS E. globulus (hardwood) than with LMS P. pinaster (softwood), but the amount of lignin removed was higher for the softwood pulp. The original milled wood samples, kraft pulps, biodelignified kraft pulps, and isolated residual lignin and milled wood lignins from the two wood samples have been characterized by pyrolysis-gas chromatography/mass spectrometry. The analysis of the pyrograms indicates that the lignin compositions of the two wood species and corresponding pulps are very different, as expected; however, the knowledge of the chemical mechanisms of delignification is very limited and requires additional work. Analytical pyrolysis is one the few degradative methods for the analysis of biopolymers that has shown a sufficient degree of success.     

Analysis of steryl esters in cocoa butter by on-line liquid chromatography-gas chromatography.

On-line liquid chromatography-gas chromatography (LC-GC) has been applied to the analysis of steryl esters in cocoa butter. Separation of the steryl esters was achieved after on-line transfer to capillary GC. HPLC removes the large amount of triglycerides and pre-separates the components of interest, thus avoiding time-consuming sample preparation prior to GC analysis. The identities of the compounds were confirmed by GC-MS investigation of the collected HPLC fraction and by comparison of the mass spectra (chemical ionization using ammonia as ionization gas) to those of synthesized reference compounds. Using cholesteryl laurate as internal standard, steryl esters were quantified in commercial cocoa butter samples, the detection limit being 3 mg/kg and the quantification limit 10 mg/kg, respectively. Only slight differences in percentage distributions of steryl esters depending on the geographical origin of the material were observed. The patterns were shown to remain unchanged after deodorization. The method described might be a valuable tool for authenticity assessment of cocoa butter.     

Gas chromatography/mass spectrometric characterisation of pyrolysis/silylation products of glucose and cellulose

The mass spectra of trimethylsilyl (TMS) derivatives of possible hydroxylated pyrolysis products of glucose and cellulose were recorded by gas chromatography/mass spectrometry (GC/MS) analyses of TMS derivatives of 2-hydroxymethylfuran, 2-hydroxy-1-methyl-1-cyclopenten-3-one, 5-(hydroxymethyl)-2-furaldehyde, 5-methyl-2-furoic acid, 4-hydroxy-6-methyl-(2H)-pyran-2-one, 2-methyl-3-hydroxy-(4H)-pyran-4-one (maltol) and 1,6-anhydro-beta-D-glucopyranose (levoglucosan, LG). Also, 2-O-TMS-1,6-anhydro-beta-D-glucopyranose, 4-O-TMS-1,6-anhydro-beta-D-glucopyranose and 2,4-bis-O-TMS-1,6-anhydro-beta-D-glucopyranose were identified from the interpretation of electron impact and chemical ionisation mass spectra of products obtained from partially silylated levoglucosan solutions, together with information from the known relative reactivities of OH groups of anhydrosugars. A peak at m/z 116 was found to be characteristic of the mass spectra of partially silylated anhydrosugars, and is absent from the mass spectra of the persilylated species. Pyrolysis/GC/MS of cellulose in the presence of hexamethyldisilazane afforded principally the 2- and 4-TMS ethers and the 2,4-bis-TMS ether of LG, whereas the 5-TMS-oxymethyl-2-furaldehyde was a prominent pyrolysis/silylation product of glucose. The mass spectra of other relevant pyrolysis/silylation products are presented.     

Palaeophytochemical Components from the Miocene‐Fossil Wood of Pinus Griffithii

Some Miocene‐fossil wood of Pinus griffithii preserved as lignified wood in brown coal was found in an open coalmine in Xundian of Yunnan Province, China. To explore its chemical components, here we show the palaeophytochemical investigation of this Pliocene‐fossil wood of P. armandii, resulting in the isolation of 11 compounds ( 1–11 ) including one new compound named 3,3‐dimethoxy‐24‐ethyl‐cholestan ( 1 ) by liquid column chromatography. Furthermore, sixteen volatiles were detected from this fossil wood by gas chromatography‐mass spectrometry (GC‐MS). These structures of 11 compounds were elucidated by analysis of their MS, 1D and 2D‐NMR spectra, and comparison with published data.     

Rapid gas chromatography/mass spectrometry determination and confirmation of patulin in apple juice

A gas chromatography/mass spectrometry (GC/MS) method was developed for the quantitative determination and confirmation of patulin extracted from apple juice. Juice is alkalized and extracted with ethyl acetate-hexane, a portion concentrated under N2, then resolubilized in acetonitrile for simple derivatization with bis(trimethylsilyl)trifluoracetamide. Patulin was determined by GC/MS using an electron-impact source and selected ion monitoring of characteristic ions. Spike levels of 20-100 microg/L gave an average recovery of 86%, and 6 ions of sample and standard spectra matched within 10% absolute for confirmation. The limits of quantitation and detection were 10 and 3 microg/L, respectively.     

Electron ionization mass spectral fragmentation of deoxynivalenol and related tricothecenes

Careful analysis of the electron impact (EI) mass spectral data obtained for the trimethylsilyl (TMS) ethers of known trichothecene mycotoxins of the deoxynivalenol group permitted the construction of a database useful for the identification of these mycotoxins directly from a gas chromatography/mass spectrometry (GC/MS) run. Structures of the ions at m/z 103, 117, 147 and 191 were elucidated by high-resolution mass spectrometry (HRMS) and a fragmentation scheme was suggested. The relative abundances of these ions in the mass spectra of the trichothecenes allowed a fast structural diagnosis during analysis of biological matrices. A new mycotoxin of this group, 3-acetylnivalenol, was tentatively identified by using MS data interpretation only.     

Analysis of anabolic steroids in hair by GC/MS/MS

A simple and sensitive gas chromatography/tandem mass spectrometry (GC/MS/MS) method is described for the detection of anabolic steroids, usually found in keratin matrix at very low concentrations. Hair samples from seven athletes who spontaneously reported their abuse of anabolic steroids, and in a single case cocaine, were analyzed for methyltestosterone, nandrolone, boldenone, fluoxymesterolone, cocaine and its metabolite benzoylecgonine. Anabolic steroids were determinate by digestion of hair samples in 1 m NaOH for 15 min at 95 degrees C. After cooling, samples were purificated by solid-phase and liquid-liquid extraction, then anabolic steroids were converted to their trimethylsilyl derivative and finally analyzed by GC/MS/MS. For detection of cocaine and benzoylecgonine, hair samples were extracted with methanol in an ultrasonic bath for 2 h at 56 degrees C then overnight in a thermostatic bath at the same temperature. After the incubation, methanol was evaporated to dryness, and benzoylecgonine was converted to its trimethylsilyl derivative prior of GC/MS/MS analysis. Results obtained are in agreement with the athletes' reports, confirming that hair is a valid biological matrix to establish long-term intake of drugs.     

Screening non-colored phenolics in red wines using liquid chromatography/ultraviolet and mass spectrometry/mass spectrometry libraries

Liquid chromatography/ultraviolet (LC/UV) and mass spectrometry/mass spectrometry (MS/MS) libraries containing 39 phenolic compounds were established by coupling a LC and an ion trap MS with an electrospray ionization (ESI) source, operated in negative ion mode. As a result, the deprotonated [M-H]- molecule was observed for all the analyzed compounds. Using MS/MS hydroxybenzoic acid and hydroxycinnamic acids showed a loss of CO2 and production of a [M-H-44]- fragment and as expected, the UV spectra of these two compounds were affected by their chemical structures. For flavonol and flavonol glycosides, the spectra of their glycosides and aglycones produced deprotonated [M-H]- and [A-H]- species, respectively, and their UV spectra each presented two major absorption peaks. The UV spectra and MS/MS data of flavan-3-ols and stilbenes were also investigated. Using the optimized LC/MS/MS analytical conditions, the phenolic extracts from six representative wine samples were analyzed and 31 phenolic compounds were detected, 26 of which were identified by searching the LC/UV and MS/MS libraries. Finally, the presence of phenolic compounds was confirmed in different wine samples using the LC/UV and LC/MS/MS libraries.     

Studies of iridoid glycosides using liquid chromatography/electrospray ionization tandem mass spectrometry

Fragmentation pathways of five iridoid glycosides have been studied by using electrospray ionization multi-stage tandem mass spectrometry (ESI-MS(n)). The first-stage MS data of the five iridoid glycosides were compared. The MS spectra showed that the adduct ions of iridoid glycosides and the formate anion were diagnostic ions to distinguish iridoid glycosides with a carboxyl group at the C-4 position or an ester group at the C-4 position. The MS fragmentation pathways of the five iridoid glycosides were also studied. Analyzing the product ion spectra of iridoid glycosides, some neutral losses were observed, such as H(2)O, CO(2) and glucose residues, which were very useful for the identification of the functional groups in the structures of iridoid glycosides. Furthermore, specific loss of one molecule of methyl 3-oxopropanoate or 3-oxopropanic acid was firstly discussed, which corresponded to the isomerization of the hemiacetal group in the structure of iridoid aglycone. According to the fragmentation mechanisms and HPLC/MS(n) data, the structures of five iridoid glycosides in a crude extract of Gardenia jasminoisdes fruit have been identified. Three compounds were compared with standards and the other two were identified as shanzhiside and genipin gentibioside by their MS(n) data without standard compounds. In order to further validate the veracity of the deduction, genipin gentiobioside was isolated from the extract of Gardenia jasminoisdes fruit using Purification Factory and was further identified by C- and H-NMR.     

Mass spectrometry of steroid glucuronide conjugates. I. Electron impact fragmentation of 5alpha-/5beta-androstan-3alpha-ol-17-one glucuronides, 5alpha-estran-3alpha-ol-17-one glucuronide and deuterium-labelled analogues

Owing to the developments of analytical instruments and interfaces (e.g. coupling high-performance liquid chromatography to mass spectrometry), there has been increased interest in new reference materials, for example in doping analysis with steroid glucuronide conjugates. The synthesized reference material has to pass several characterization steps including the use of gas chromatography/mass spectrometry (GC/MS) for its structure confirmation. In the present study, the fragmentation and mass spectrometric behaviour of several steroid glucuronide conjugates of endogenous and anabolic steroids after derivatization to pertrimethylsilylated products and to methyl ester pertrimethylsilylated products were investigated using GC/MS ion trap and GC/MS quadrupole instruments. The mass spectra of the derivatives of androsterone glucuronide, d5-androsterone glucuronide, epiandrosterone glucuronide, etiocholanolone glucuronide, 11beta-hydroxy etiocholanolone glucuronide, 19-norandrosterone glucuronide, d4-19-norandrosterone glucuronide and 1alpha-methyl-5alpha-androstan-3alpha-ol-17-one glucuronide are presented and the origin of typical fragment ions of the glycosidic and steroidal moieties is proposed, based on different derivatization techniques including derivatization with d18-bistrimethylsilylacetamide, methyl ester and trimethylsilyl ester derivatization and selected reaction monitoring. Typical fragmentation patterns which are related to the steroid structure are discussed.     

Identification of Abnormal Urinary Organic Acids in Inherited Metabolic Diseases by Gas Chromatography-Mass Spectrometry

A gas chromatography/mass spectrometry (GC/MS) coupled system has been established for the confirmatory identification of abnormal urinary organic acids in inherited metabolic diseases. Samples of patient urines were extracted with an organic solvent and trimethylsilylated (TMS). A mass spectra of gas chromatographically separated TMS derivatives can be obtained using the GC/MS coupled system with a single analytical run. Those compounds with close methylene units (e.g., 4-hydroxyphenylacetaic acid and phenylpyruvic acid) in the gas chromatograph can be identified by their specific mass spectra. The results indicate that this GC/MS system is a powerful method for identifying abnormal urinary organic acids. These acids can be identified by comparison with authentic mass spectra established in our laboratories or with mass spectra files from other sources or they can be directly identified by analysis of the mass spectrum. By using this system, we were able to make positive identification of several inherited metabolic diseases found in Chinese patients, including phenylketonuria, propionic acidemia, and methylmalonic aciduria. This GC/MS system is a powerful tool for the diagnosis of inherited metabolic diseases.     

Deuteration as an aid to the high-temperature gas chromatography—mass spectrometry of steryl fatty acyl esters

A method is presented for reducing degradative losses of steryl fatty acyl esters bearing polyunsaturated fatty acyl moieties during high-temperature gas chromatography (GC) and combined high-temperature GC—mass spectrometry (MS). The method employs selective deuteration of the double bonds in the fatty acyl moiety using a homogeneous catalyst (Wilkinson's catalyst). The Δ⁵ double bond, which occurs in the most commonly occurring plant and animal sterols, is not deuterated under the conditions described. In addition to improving GC behaviour, the method has the advantage of preserving structure information by labelling each double bond present in the original unsaturated fatty acyl moiety. The carbon number and degree of unsaturation of the labelled fatty acyl moiety are readily revealed by combined GC—MS employing negative-ion ammonia chemical ionisation. Two applications of the method are demonstrated in the analysis of steryl fatty acyl esters isolated from a rape seed oil and ovary tissue of the marine prawn Penaeus monodon.