GC-SIMS

GC-MS and GC-AED techniques were used for the direct analysis of used tyre vacuum pyrolysis oil [255]. Antioxidants and antiwear additives (0.25-5 wt% DODPA, a-NPA, TCPs, TPP, IPPs) in lubricating synthetic oils, essentially esters of branched-chain alcohols such as pentaerythritol, neopentylglycol and trimethylolpropane, were determined by means of GC-SIM-MS using diphenylamine (DPA) as an internal standard [256] similarly, TCPs, TPP, IPPs, DPs and I2P were quantitatively analysed by GC-FPD using triethylphosphate (TEP) as an internal standard. RSD values of 3-6% were reported for GC-SIM-MS, and 7-9 % for GC-FPD. 

A sensitive method for primary amines is shown in reaction 2, leading to the corresponding 7V-benzenesulfonyl-/V-trifluoroacetyl derivatives. These can be determined by GC-ECD using SE-30 columns LOD 1-5 pg, which is about 200 times more sensitive than GC-FID. The method was applied for determination of phenethylamine (33) in urine110. This analysis was performed also by LLE into n-pentane, derivatization to the benzenesulfonamide and GC-FPD using a capillary column recoveries of aliphatic primary amines in urine were 91-107%, RSD 0.2-4.5%111,112. Amines in environmental waters and sediments were determined after LLE with dichloromethane, derivatization with benzenesulfonyl chloride and GC-SIM-MS LOD 0.02-2 pg/L of water and 0.5-50 ng/g of sediment113. 

Gas chromatography-selected ion monitoring (GC-SIM) is often used for the quantification of GAs by GC-MS, an internal standard labeled with stable isotopes (usually with deuterium) being used as the most reliable and sensitive method. A mass chromatogram reconstructed from full-scan GC-MS is also used for semiquantification. The amounts of GAs are determined by measuring the peak areas of ions characteristic for each GA and comparing these areas to those of authentic samples. When an internal standard labeled with a stable isotope is used, the ratio of the area of an ion peak characteristic for a sample and the area of the corresponding peak of the labeled internal standard is used for quantification. The analysis of GAs by GC-MS has been discussed in numerous publications and review articles.254-256... 

The A,A-dimethylaminomethylene derivatives (81) of ten HAA listed in Table 2.B-C were analyzed by GC-MS or GC-NPD. All mass spectra showed the [M]+ peak. More convenient was analysis with NPD for which the LOD for injection were in the range of 2 pg for 25a to 15 pg for 23a, with good linearity from 0.5 to 10 ng in all the studied cases170. This method was used in detection and estimation of 25a, 28b and 30a in the Danube River106. A routine search for the carcinogenic HAA of Table 2.B-C in meat and process flavor products was proposed, based on the cleanup methods for such samples mentioned in Section III.A.5, derivatizing with 3,5-bis(trifluoromethyl)benzyl bromide (82), and capillary GC-SIM-ESI-MS143. 

Microanalytical techniques, consisting of methaneboronation of the vicinal hydroxyls ( bismethaneboronate or methaneboronate-TMS-ether ) have been developed by Takatsutoet al. for the GC/MS or GC/SIM of brassinosteroids (16). Consequently brassinone (15), 24-ethylbrassinone (12) and 28-norbrassinolide (14) were detected by GC/MS without isolation from Chinese cabbage ( Brassica campestris ), green tea ( Thea sinensis ) and chestnut insect galls ( Distilium racemosum ) (17-19). The presence of brassinolide and castasterone in the tissues of these plants has been also detected. 

A combination of the rice lamina inclination assay and GC/MS or GC/SIM analyses have allowed for identification of the brassinosteroids in various plant tissues These include brassinolide, 6-deoxodihydrocastasterone, brassinone from the insect galls of Castanea crenata (18,20) castasterone and 6-deoxodihydrocastasterone from the stems, leaves and flowers of Castanea crenata (20) brassinolide, castasterone, typhasterol (7), and teasterone (8) from leaves of Thea sinensis (21-23) castasterone and brassinone from the fruit of Pharbitis purpurea ( Japanese morning glory ) (24) brassinolide, castasterone and 24-epibrassinolide(29) from immature seed and/or pollen of Vida faba (broad bean ) (25,26) brassinolide and castasterone from the pollen of Alnus glutinosa (European alder) (27). 

The extract of cells of Catharanthus roseus were partially purified by reverse phase HPLC and were analyzed by GC-MS, disclosing that the major brassinosteroids in the cells are brassinolide and castasterone (5). Change of the contents of brassinolide and castasterone during cell growth was pursued by GC-SIM using D brassinolide and... 

In order to understand the growth retardation mechanism of S-uniconazole, the shoots of Pisum sativum L. treated with S- and R-uniconazoles were analyzed in terms of the levels of the endogenous GAs, BRs, and phytosterols. Only referring to BRs, it is of interest to examine whether uniconazoles modify the biosynthesis of BRs. BRs contained in the shoots of P. sativum L. were extracted, purified, and analyzed by the GC/MS. GC/MS analysis of the active fraction led to the identification of CS m/z (rel. int.) 512 (M+, 54%), 155 (100%). GC/SIM quantitation using an internal standard (d6-CS) revealed that the content of CS in the control plants was 0.9 ng/g fir. wt. and, after treatment with S- and / -uniconazoles, reduced to 54% and 34% of the controls, respectively. The result suggests that the altered metabolism of BRs is likely to be involved in the action mechanism of S-uniconazole. 

Fauhl, C. and Wittkowski, R. (1992). Determination of ethyl carbamate in wine by GC-SIM-MS after continous extraction with diethyl ether,/. High. Res. Chromatogr., 15,203-205. 

Detection of a target analyte may be tedious with GC-SIM because signal interference may conceal the signal. A method for mass spectrum extraction of GC-MS data has been incorporated into an automated software program developed by the National Institute of Standards, called Automated Mass Spectral Deconvolution and Identification system (AMDIS). The LEGO corporation has also developed a software program used to resolve overlapped signals from GC-TOF-MS. 

GC-SIM gas chromatography-selected ion monitoring MTBSTFA Al-methyl-Al-r- butyldimethylsilyltrifluoroacetamide... 

Details of numerous HPLC methods that can be used for the purification of plant hormones are presented in Rivier and Crozier [1]. With some tissues, partitioning, cartridge systems and/or immunoaffinity chromatography can provide adequate sample purification prior to ABA and lAA analysis. However, HPLC fractionation is almost always required before GC-SIM analysis of individual cytokinins and GAs. As illustrated in Figs 2 and 3, good separations of free GAs and cytokinins of wide ranging polarity can be obtained by gradient elution, reverse phase HPLC. 

As mentioned in the Section 1, physico-chemical methodology for quantitative analysis of plant hormone focuses primarily on GC-SIM, although HPLC with selective fluorescence detection continues to be used for lAA analysis in some laboratories. Procedures, such as the 2-methylindolo-a-pyrone assay for lAA analysis [82], are now rarely utilised. With the exception of ethylene quantification [2] there is little use of non-MS-based GC detection techniques, despite the fact that selective analysis at the picogram level is achieved for ABA with an electron capture detector [83], and lAA and cytokinins with a nitrogen phosphorus detector [84,85]. The reason for the demise of these GC procedures is that the detectors are destructive and this precludes the reliable recovery of labelled internal standards for radioassay and isotopic dilution analysis. The usual compromise was to take two aliquots of the purified samples, one for GC analysis and the other for the determination of radioactivity. The accuracy of this approach is dependent upon the questionable assumption that the radioactivity in the purified sample is associated exclusively with the compound under study. In an attempt to circumvent this problem, a double standard isotope dilution procedure was devised for the quantitative analysis of lAA in which one internal standard was used to correct for losses during sample preparation and a second for GC quantification [86]. This procedure was used in several... 

Although quantitative analysis of endogenous plant hormones by traditional GC has serious limitations, isotopic dilution analysis by GC-SIM using a single internal standard labelled with a stable isotope, such as H, C or N, is a completely different proposition [3-6]. Because the cost of a simple, computer-controlled, quadrupole-mass spectrometer has fallen substantially, and many highly enriched, isotopically-labelled compounds suitable for use as internal standards in quantitative analysis, can be either synthesized (1) or purchased from commercial sources (see Table 1), capillary GC-SIM is now the quantitative assay of choice in the vast majority of laboratories in which endogenous plant hormones are analysed on a routine basis. 

Fig. 4. Calibration curve for isotope dilution analysis of GA by capillary GC-SIM using [ H lCAjo as an internal standard [Moritz and Nilsson, unpublished data].

It has been noted that HPLC can result in a slight separation of deuterated compounds, labelled with more than two H atoms, from their probated equivalents [92]. Thus,when HPLC is used for sample purification, broad rather than narrow fractions should be collected for further analysis. Deuterated compounds also have slightly shorter retention times than their Ho-labelled analogues on columns of low or medium polarity but this has no significant effect on the performance of GC-SIM in isotopic dilution analysis. However, calculations of isotopic dilution based on full-scan data require spectral averaging [12]. 

Fig. 5. Capillary GC-SIM of a 1/10 aliquot of a methylated and trimethylsilylated semi-purified extract from 5 g of Populus tremulaxtremoluides leaves. Traces normalised to 100% for the most intense ion. Internal standard 10 ng [ HjJGAjo. [Moritz and Nilsson, unpublished data].

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