Edmond de Hoffmann

Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry

Eleven naturally occurring flavonoid aglycones, belonging to the representative flavone, flavonol, and flavanone types were separated by high performance liquid chromatography and analyzed on-line with negative ion electrospray ionization tandem mass spectrometry (ESI-MS/MS). In order to resolve the MS/MS spectra obtained, each compound was reinvestigated by direct loop injections using an ion trap mass spectrometer. The MSn spectra obtained allowed us to propose plausible schemes for their fragmentation supported by the analysis of five complementary synthetic flavonoid aglycones. The negative ion ESI-MS/MS behavior of the different aglycones investigated in this study revealed interesting differences when compared with the previously described patterns obtained using various ionization techniques in positive ion. Thus, concerning the retro Diels-Alder (RDA) fragmentation pathways, several structurally informative anions appeared highly specific of the negative ion mode. In addition, a new lactone-type structure, instead of a ketene, was proposed for a classic RDA diagnostic ion. We also observed unusual CO, CO2, and C3O2 losses which appear to be characteristic of the negative ion mode. All these results and these unusual neutral losses show that the negative ion mode was a powerful complementary tool of the positive ion mode for the structural characterization of flavonoid aglycones by ESI-MS/MS.

Tandem mass spectrometry: A primer

Tandem mass spectrometry (MS/MS) employs two stages of mass analysis in order to examine selectively the fragmentation of particular ions in a mixture of ions. The various types of instruments which can be used to perform this experiment are described, including those based on separation of the mass-analysis events in time and those based on measurements in physically separate analysers. The several MS/MS scan types-product scans, precursor scans and neutral loss scans-are presented and examples of their applications are provided. These include the characterization of individual compounds and the recognition of groups of compounds with specified functional groups. Applications are shown to the determination of compounds present in complex mixtures. The improvement in signal-to-noise ratio achieved by MS/MS when compounds are ionized by methods which produce high chemical noise is illustrated for choline derivatives. Fundamental aspects of collisional activation in the low and high collision energy range are reviewed.

Variation of DIMBOA and related compounds content in relation to the age and plant organ in maize.

We report the variation of all 1,4-benzoxazin-3-one derivatives content detectable in maize with plant age in roots and aerial parts. Our results show that the concentration of hydroxamic acids, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc) and its 8-methoxylated analogue (DIM2BOA-Glc) is high after seed germination and then decreases with plant age. Nevertheless, these compounds continue to be biosynthesised during 6-10 days after germination. Variation in concentration of N-O-methylated DIMBOA-Glc (HDMBOA-Glc) is similar to the one of hydroxamic acids in aerial parts. On the contrary, in roots, its concentration remains relatively stable with plant age. After 10 days, HDMBOA-Glc becomes the main compound in roots. This compound is also present in higher concentration than hydroxamic acids in the oldest leaf of 20-day-old maize. The presence of four other DIMBOA related compounds in maize plants depends on variety, age and tissue. The role of these compounds in plant resistance to aphids is discussed.

Collision-induced dissociation of alkali metal cationized and permethylated oligosaccharides: Influence of the collision energy and of the collision gas for the assignment of linkage position

Tandem mass spectrometry has been used to study the collision-induced decomposition of [M+Na]+ ions of permethylated oligosaccharides. It is shown that many linkage positions in one compound may be determined by the presence or absence, in a single spectrum, of specific fragment ions that arise from the cleavage of two ring bonds and that the yield of such ions depends strongly on the collision energy and nature of the collision gas. In contrast to the behavior of monolithiated native oligosaccharides, the collision-induced decomposition of the sodiated and permethylated oligosaccharide samples at low energy leads to preferential cleavage of glycosidic linkages. At high collision energies, the fragment ions formed by cleavage of more than one bond are greatly enhanced, especially when helium is replaced by argon as the collision gas. Furthermore, argon is the more efficient collision gas in inducing fragmentation of the precursor ions. As an example of the application of this method, the discrimination between the 1 → 3 and 1 → 6-linked mannose residues in the common core of N-glycans is described.

Non-injured maize contains several 1,4-benzoxazin-3-one related compounds but only as glucoconjugates

DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) and its derivatives are known to participate in the resistance of the Poaceae to several pests. A more efficient analytical method for the determination of these compounds is reported, employing high performance liquid chromatography coupled with UV and atmospheric pressure chemical ionisation-tandem mass spectrometric detection. The results showed that in non-injured 5 or 15-day-old plants only the glucosylated forms of DIMBOA and related compounds were present: aglycones were only detected when enzymatic deglucosylation occurred during sample work-up. Maize plants contain seven glucosides related to DIMBOA belonging to three classes: hydroxamates, lactams and N-O-methylated hydroxamate derivatives. Rarely reported dimethoxylated compounds and 8-methoxy-N-O-methylated DIMBOA are now reported in the Poaceae, the latter for the first time. Their presence and relative concentrations in 5 and 15-day-old maize roots and aerial parts are discussed. The non-hydroxamic N-O-methylated DIMBOA is present in the plant in significant quantities which exceed all others in 15-day-old maize roots. After enzymatic degradation, deglucosylation products from hydroxamates and lactams are observed. The N-O-methylated deglucosylated compounds are not observed as they are chemically degraded, most probably to benzoxazolinone derivatives. The results reported here provide a new view on the DIMBOA-related compounds present in maize plants, and the implications of these findings are discussed. Copyright (C) 1999 John Whey & Sons, Ltd.

Study on in vivo and in vitro metabolism of dimethylformamide in male and female rats

The study of dimethylformamide (DMF) metabolism by rat tissues in vitro indicates that formaldehyde is not a metabolic product as previously reported [1]. Furthermore, no other monocarbon derivative (CO, CH3OH, HCOOH, CH4) was detected when DMF was incubated with a fortified liver preparation. One metabolic product is methylhydroxymethylformamide (DMF-OH) measured as N-methylformamide (NMF) due to the breakdown of the hydroxymethyl group during gas chromatography. It was usually believed that the main metabolite excreted in urine following administration of DMF to male and female rats was NMF. The results of this study indicate that DMF-OH constitutes the main metabolite in vivo. A quantitatively less important urinary metabolite, hydroxymethylformamide (NMF-OH), is determined as formamide (F) by gas chromatography. In male and female rats, partial hepatectomy reduces markedly the in vivo biotransformation of DMF. Following administration of DMF or NMF, the total amount of metabolites (DMF-OH and/or NMF-OH) excreted in urine is identical in both sexes, but female rats excrete more unchanged parent compound than male rats. The rate of NMF-OH excretion in urine following high doses of DMF supports the hypothesis that DMF may inhibit its own biotransformation.

On the Presence of Phosphorylated Sphingoid Bases in Rat-tissues - a Mass-spectrometric Approach

A simple and straightforward procedure to analyze phosphorylated sphingoid bases has been developed. After phase separation of lipid extracts under alkaline conditions, the compounds were quantitatively recovered in the aqueous upper phase. Following a clean‐up of the aqueous phase on C18‐solid phase extraction columns, the amino‐group of the bases was derivatized by means of phenylisothiocyanate addition. FAB‐MS of the phenylthiocarbamate derivatives of sphingenine‐ and sphinganine‐phosphate in the negative mode revealed the expected pseudo‐molecular ions (M‐1) at 513 m/z and 515 m/z, respectively. Moreover, a typical fragmentation pattern, characterized by the loss of the phenylthiocarbamate moiety (m/z = 135), was observed. When applied to rat tissues, the presence of sphingenine‐phosphate in brain, kidney and liver could easily be demonstrated. Highest levels, amounting to 5 nmol/g of wet weight, were present in brain.

Fragmentation of conjugate bases of esters derived from multifunctional alcohols including triacylglycerols

Enolate anions of esters from 1,2 and 1,3 diols undergo an internal nucleophilic substitution reaction that produces a β-ketoester and an alkoxide ion within the molecular species. These intermediate ions undergo two competitive fragmentation pathways. The first pathway corresponds to a second nucleophilic substitution of the ketoester by the alkoxide that yields a neutral cyclic ether and the β-ketoacid carboxylate. The latter then loses carbon dioxide and produces the enolate anion of the corresponding ketone. The second proposed pathway is stepwise: it starts with a proton transfer from the methylene group between the two carbonyls to the alkoxide anion that produces an alcohol and the enolate ion of the β-ketoester inside the molecular species. The latter undergoes cleavage of the ester bond induced by the negative charge to yield an ion-dipole complex composed of a neutral acylketene and an alkoxide ion. The direct dissociation of this ion-dipole complex competes with an internal proton exchange to yield a new complex that consists of an alcohol molecule and the anion of the acylketene, which can also dissociate. The fragmentation pathway that leads to the ketone enolate is sensitive to the relative positions (1,2 or 1,3) of the esters on the molecular backbone. This position-sensitive reaction is useful for the assignment of the primary and secondary positions in triacylglycerols, even in mixtures, as shown by some examples.

Faster identification of mycobacteria using gas liquid and thin layer chromatography

Gas liquid chromatography (GLC) and thin layer chromatography (TLC) analysis of cell wall content was used for identification of mycobacteria isolated in primary cultures. GLC permitted determination of the fatty acid and alcohol profiles ofMycobacterium simiae andMycobacterium marinum and detection of a peak inMycobacterium ulcerans formerly described forMycobacterium malmoense. Using the data obtained to fill some of the gaps in the dichotomic trees of Tisdall et al. and Jantzen et al., GLC analysis allowed full identification of 8 of 22 mycobacterial species after 24 hours. The other 14 species could be divided into four groups on the basis of similar findings on GLC. TLC was used for full identification of three species. The identification results of conventional methods were concordant with those of GLC and TLC in 161 of 169 strains (93 %) representing 21 different species. Using primarily chromatography for analysis of cell wall content, and in the case of some species complementary biochemical tests, the identification procedure could be shortened to a maximum of three days after primary culture.

Fast-atom bombardment mass spectrometry and low energy collision-induced tandem mass spectrometry of tauroconjugated bile acid anions

Fragmentation of negative ions produced by fast-atom bombardment (FAB) from 14 tauroconjugated bile acids and some of their deuterated analogs has been studied by mass spectrometry and by collision-induced dissociation (CED) tandem mass spectrometry at low energy.Low energy collision-induced dissociation of the deprotonated molecules [M - H]− of these tauroconjugated bile acids leads to both charge-driven and charge-remote fragmentations (CRF). The former yields neutral loss from the side chain with charge migration during the fragmentation process. These fragments dominate the CID spectra, but are absent from the FAB spectra. Their relative abundances are dependent on the number and the positions of the hydroxyl groups in the steroid nucleus and thus permit distinction among some positional isomers.The CRF fragments correspond to cleavages in the side chain up to fragmentations across the steroid rings with charge retention on the sulfonate group. These CRF fragments, which also are useful for structural identification, are less intense in CID than in FAB spectra. It appears that these charge-remote fragments are favored by unsaturation in the steroid rings, either as keto groups or as endocyclic double bonds. Tandem mass spectrometry combined with the use of deuterated analogs demonstrates that the structures of the survivor pseudomolecular ions and of the CRF fragments are not rearranged.