Identification of 1,4-Benzoxazin-3-ones in Maize Extracts by Gas-Liquid Chromatography and Mass Spectrometry

Gas-liquid chromatography-mass spectrometry (GLC-MS) has been used for the separation, detection, and identification of 1,4-benzoxazin-3-ones (hydroxamic acids and lactams) and benzoxazolinones found in maize (Zea mays L.) extracts. Compounds of interest were partitioned into ethyl acetate from aqueous maize seedling extracts. For analysis by GLC-MS, trimethylsilyl derivatives were prepared, chromatographed on a column of 3% OV-1, and detected in the mass spectrometer. Mass spectra were obtained for all peaks present in extracts of four maize lines. A data comparison system was developed for relating unidentified spectra to the spectra of the reference compounds. Based on spectral comparisons, three hydroxamic acids (2,4-dihydroxy-2H-1, 4-benzoxazin-3(4H)-one; 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one; and 2,4-dihydroxy-7,8-dimethoxy-2H-1,4-benzoxazin-3(4H)-one), three lactams (2-hydroxy-2H-1,4-benzoxazin-3(4H)-one; 2,7-dihydroxy-2H-1,4-benzoxazin-3(4H)-one; and 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one), one benzoxazolinone (6-methoxybenzoxazolinone), and two organic acids (malic and aconitic) were identified in the extracts. In addition, one other hydroxamic acid and one other related compound were tentatively identified based on mass spectral evidence.     

Natural Variation in Maize Aphid Resistance Is Associated with 2,4-Dihydroxy-7-Methoxy-1,4-Benzoxazin-3-One Glucoside Methyltransferase Activity

Plants differ greatly in their susceptibility to insect herbivory, suggesting both local adaptation and resistance tradeoffs. We used maize (Zea mays) recombinant inbred lines to map a quantitative trait locus (QTL) for the maize leaf aphid (Rhopalosiphum maidis) susceptibility to maize Chromosome 1. Phytochemical analysis revealed that the same locus was also associated with high levels of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) and low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). In vitro enzyme assays with candidate genes from the region of the QTL identified three O-methyltransferases (Bx10a-c) that convert DIMBOA-Glc to HDMBOA-Glc. Variation in HDMBOA-Glc production was attributed to a natural CACTA family transposon insertion that inactivates Bx10c in maize lines with low HDMBOA-Glc accumulation. When tested with a population of 26 diverse maize inbred lines, R. maidis produced more progeny on those with high HDMBOA-Glc and low DIMBOA-Glc. Although HDMBOA-Glc was more toxic to R. maidis than DIMBOA-Glc in vitro, BX10c activity and the resulting decline of DIMBOA-Glc upon methylation to HDMBOA-Glc were associated with reduced callose deposition as an aphid defense response in vivo. Thus, a natural transposon insertion appears to mediate an ecologically relevant trade-off between the direct toxicity and defense-inducing properties of maize benzoxazinoids.     

Determination of Chloropicrin in Fumigants by Gas-Liquid Chromatography

The present time, chloropicrin (CP) is commonly used as the fumigant for stored grain insects, nematodes, soil fungus and weed seeds. The quantitative determination of CP has been performed by total chlorine method¹⁾, colorimetric method²⁾, and polarography³⁾. However, these methods require a great deal of trouble. In the previous paper, the author reported on the operating conditions and retention datas for the determination of CP by gas-liquid chromatography (GLC)⁴⁾. The method reported here is an application of this work for analysis of liquid soil fumigants.     

Identification of Vibrio cholerae by Pyrolysis Gas-Liquid Chromatography

Cholera-like vibrios examined by pyrolysis gas-liquid chromatography could be distinguished from other common aerobic gram-negative bacilli, including oxidase-positive organisms, e.g., Aeromonas. Vibrios in Heiberg group I were subdivided into three types on the basis of differences in one complex in the chromatogram, and these closely corresponded with the identification as classical, El Tor, or "intermediate" biotypes of Vibrio cholerae by conventional methods.     

Detection of Griseofulvin and Dechlorogriseofulvin by Thin-Layer Chromatography and Gas-Liquid Chromatography

A rapid and accurate method is described for the determination of griseofulvin and dechlorogriseofulvin extracted from Penicillium urticae with chloroform. Thinlayer chromatography was used to tentatively identify griseofulvin or dechlorogriseofulvin, or both. Two gas-liquid chromatographic systems provided additional qualitative information and simultaneous quantitation of the individual compounds.     

Determination of Pyrrolnitrin and Derivatives by Gas-Liquid Chromatography

A gas-liquid chromatographic technique was applied to the separation of pyrrolnitrin and its derivatives. The simultaneous use of a flame detector and an electron capture detector made possible the distinction between the nitro derivatives of pyrrolnitrin and the other metabolites. The metabolites could be readily quantitated with the electron capture detector, offering a much more sensitive assay than the flame detector.     

Biosynthesis of 1,4-benzoxazin-3-ones in Zea mays

¹⁴C- and ¹⁵N-anthranilic acid are incorporated into the 1,4-benzoxazin-3-ones in maize seedling leaves with low dilution of the isotope. o-Aminophenol and 3-hydroxyanthranilic acid are not incorporated and are probably not intermediates. The cyclic hydroxamic acid and lactam members of the 1,4-benzoxazin-3-one group of compounds are readily interconverted.     

Biotransformation of 2-Benzoxazolinone and 2-Hydroxy-1,4-Benzoxazin-3-one by Endophytic Fungi Isolated from Aphelandra tetragona

The biotransformation of the phytoanticipins 2-benzoxazolinone (BOA) and 2-hydroxy-1,4-benzoxazin-3-one (HBOA) by four endophytic fungi isolated from Aphelandra tetragona was studied. Using high-performance liquid chromatography-mass spectrometry, several new products of acylation, oxidation, reduction, hydrolysis, and nitration were identified. Fusarium sambucinum detoxified BOA and HBOA to N-(2-hydroxyphenyl)malonamic acid. Plectosporium tabacinum, Gliocladium cibotii, and Chaetosphaeria sp. transformed HBOA to 2-hydroxy-N-(2-hydroxyphenyl)acetamide, N-(2-hydroxyphenyl)acetamide, N-(2-hydroxy-5-nitrophenyl)acetamide, N-(2-hydroxy-3-nitrophenyl)acetamide, 2-amino-3H-phenoxazin-3-one, 2-acetylamino-3H-phenoxazin-3-one, and 2-(N-hydroxy)acetylamino-3H-phenoxazin-3-one. BOA was not degraded by these three fungal isolates. Using 2-hydroxy-N-(2-hydroxyphenyl)[¹³C₂]acetamide, it was shown that the metabolic pathway for HBOA and BOA degradation leads to o-aminophenol as a key intermediate.     

Induction and detoxification of maize 1,4-benzoxazin-3-ones by insect herbivores

In monocotyledonous plants, 1,4‐benzoxazin‐3‐ones, also referred to as benzoxazinoids or hydroxamic acids, are one of the most important chemical barriers against herbivores. However, knowledge about their behavior after attack, mode of action and potential detoxification by specialized insects remains limited. We chose an innovative analytical approach to understand the role of maize 1,4‐benzoxazin‐3‐ones in plant–insect interactions. By combining unbiased metabolomics screening and simultaneous measurements of living and digested plant tissue, we created a quantitative dynamic map of 1,4‐benzoxazin‐3‐ones at the plant–insect interface. Hypotheses derived from this map were tested by specifically developed in vitro assays using purified 1,4‐benzoxazin‐3‐ones and active extracts from mutant plants lacking 1,4‐benzoxazin‐3‐ones. Our data show that maize plants possess a two‐step defensive system that effectively fends off both the generalist Spodoptera littoralis and the specialist Spodoptera frugiperda. In the first step, upon insect attack, large quantities of 2‐β‐d ‐glucopyranosyloxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one (HDMBOA‐Glc) are formed. In the second step, after tissue disruption by the herbivores, highly unstable 2‐hydroxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one (HDMBOA) is released by plant‐derived β‐glucosidases. HDMBOA acts as a strong deterrent to both S. littoralis and S. frugiperda. Although constitutively produced 1,4‐benzoxazin‐3‐ones such as 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one (DIMBOA) are detoxified via glycosylation by the insects, no conjugation of HDMBOA in the insect gut was found, which may explain why even the specialist S. frugiperda has not evolved immunity against this plant defense. Taken together, our results show the benefit of using a plant–insect interface approach to elucidate plant defensive processes and unravel a potent resistance mechanism in maize.     

Virulence Prediction of Yersinia enterocolitica by Pyrolysis Gas-Liquid Chromatography

[This corrects the article on p. 648 in vol. 40.].     

Indole-3-acetic Acid in Douglas Fir: Analysis by Gas-Liquid Chromatography and Mass Spectrometry

We sought evidence for the occurrence and seasonal variation of indole-3-acetic acid (IAA) in shoots of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco).     

Identification of Abscisic Acid in Tulipa gesneriana L. by Gas-Liquid Chromatography with Electron Capture and Combined Gas-Liquid Chromatography and Mass Spectrometry

A major growth inhibitory substance of tulip bulbs (Tulipa gesneriana L. cv Paul Richter) has been unequivocally shown to be abscisic acid (ABA). The ABA methyl ester of the free ether-soluble acid fractions of tulip organs had the identical retention time on gas-liquid chromatography with electron capture detector as authentic ABA methyl ester. In addition, the mass spectra were the same. On a unit dry matter basis, the basalplate and floral shoot contained 3.6 and 2.6 times more ABA than the fleshy scales, respectively.     

Determination of Poly-beta-Hydroxybutyrate and Poly-beta-Hydroxyvalerate in Activated Sludge by Gas-Liquid Chromatography

A convenient gas-liquid chromatography procedure to quantify poly-beta-hydroxybutyrate and poly-beta-hydroxyvalerate in activated sludge was developed by combining lyophilization of the samples, purification of the chloroform phase by water reextraction, and the use of capillary columns. With a flame ionization detector the sensitivity was estimated at 10 g/liter.     

Determination of Steam-Volatile Organic Acids in Fermentation Media by Gas-Liquid Chromatography

Five gas chromatographic liquid phases (25% Carbowax 20 M plus 4% H₃PO₄, 17.5% dioctyl sebacate plus 7.5% sebacic acid, 17.5% dioctyl sebacate plus 7.5% docosanoic acid, 5% Tween 80, and 20% LAC-296 [poly (diethylene glycol adipate)] plus 2% H₃PO₄) were studied with respect to their utility in the separation and quantitation of steam-volatile organic acids commonly produced in fermentation. Optimal operating conditions and column stability for routine analysis were established. An Aerograph Hy-Fi gas chromatograph was used for all work, except the studies with Tween 80 in which an Aerograph A-90-C was employed. Chromatographic traces are presented of volatile fatty acid analyses with each of the liquid phases. Complete separation of all isomers of the fatty acids from C₂ to C₅ was accomplished by the Carbowax 20 M plus H₃PO₄, dioctyl sebacate plus sebacic acid, and dioctyl sebacate plus docosanoic acid columns. The latter two liquid phases were extremely unstable and proved to be unsatisfactory for analysis of aqueous samples. A column of Carbowax 20 M + H₃PO₄ separated steam-volatile organic acids completely. The volatile fatty acid isomers were separated by 5% Tween 80 somewhat less completely, and the peak shapes were not as sharp and symmetrical as that desired for good quantitative work. LAC-296 (20%) plus 2% H₃PO₄ proved to be the most satisfactory of the liquid phases for routine analysis of deproteinated in vitro rumen fermentation media. The column has been used for routine analysis of rumen fermentation fluid and in vitro rumen incubation fluid. All the organic acids from C₂ to C₅, except isobutyric, could be quantitated with this column. Stability of the column with the aqueous solutions was extremely good. The standard deviation of the analysis of each volatile acid component in a fermentation fluid was less than 0.5 molar per cent. The short-chain organic acids (C₂ to C₅) were shown to be extremely stable in aqueous solution for as long as 6 months after preparation for gas chromatographic analysis by protein precipitation with metaphosphoric acid-H₂SO₄ and refrigeration at 4 C in stoppered tubes.     

Lipid Patterns of Selected Microorganisms as Determined by Gas-Liquid Chromatography

The cellular lipid patterns of seven strains of microorganisms were examined by gas-liquid chromatography in this preliminary study. The chloroform methanol-soluble lipids were extracted by the Soxhlet method from dried cultures which had been grown at 25 +/- 2 C for 18 hr with mechanical shaking. The cellular extract was methylated by use of a low temperature sulfuric acid method, and the resulting methyl esters were chromatographed. Considerable differences in the lipid patterns among the seven microorganisms tested indicated that this method might be useful for the identification of closely related microbial genera, and possibly for species differentiation.     

Gas-Liquid Chromatographic Analysis of Indole-3-acetic Acid Myoinositol Esters in Maize Kernels

An improved method of fractionating the myoinositol esters of indoleacetic acid (IAA) from maize kernels by gas-liquid chromatography has been developed. Mass spectrometry was employed as an aid in identification of the esters. Maize kernels contain three groups of esters of IAA: (a) IAA myoinositols, (b) IAA myoinositol arabinosides, and (c) IAA myoinositol galactosides. Each group has three chromatographically distinguishable isomers. The glycosylinositols described are unique in that carbon 1 of the sugar is attached to the hydroxyl at C-5 of the myoinositol.     

Analysis of Streptococcal Cell Wall Fractions by Curie-Point Pyrolysis Gas-Liquid Chromatography

A streptococcal strain, classified as Z(3)III was differentiated from its mutant strain, Z(3), lacking the type III polysaccharide antigen, by Curie-point pyrolysis gas-liquid chromatography. Differences observed in pyrograms of whole cells or cell envelopes of both strains could be directly related to the pyrolysis pattern of the purified type III antigen. The same results were obtained when streptococcus F III and its mutant were analyzed. Whereas the pyrolysis patterns of the type III antigen extracted from Z(3)III and F III bacteria were identical, marked differences were found in pyrograms of the serologically identical type III antigen isolated from the culture medium. Type III antigen was also easily differentiated from the purified type I, II and IV antigens. From the above findings it was concluded that pyrolysis gas-liquid chromatography can be used as a tool for the quality control and identification of streptococcal cell wall components.     

Separation and Quantitation of Neomycins as Free Base by Paper Chromatography

Neomycin B and C and neamine were separated as free bases in the solvent system methyl ethyl ketone-tert-butyl alcohol-methanol-6.5 N NH₄OH (16:3:1:6 v/v) and quantitatively estimated.     

Reduction of nitro-substituted 1,2-dihydro-3H-1,4-benzodiazepine- 2-ones by E. coli cells immobilized in carrageenan

Reduction of nitro-substituted 1,2-dihydro-3H-1,3-benzodiazepine-2-ones by E. coli cells immobilized in carrageenan was studied. The corresponding amines are the sole products with a 100% yield as compared to the native cells. Conditions for immobilization of E. coli cells in the home-produced carrageenan was worked out: the cell to carrageenan ratio is 1:10 (w/w), granulation in toluene at 0-(+)4 degrees, treatment with 0.3-0.4 M KCl. The carrageenan-immobilized cells are stable upon storage, repeated usage (after 10 cycles about 80% of the initial activity is retained), and when being used in column fermenters.