Gas Chromatography-mass Spectrometry-selected Ion Monitoring Research Articles

Implication of Gibberellins in Head Smut (Sporisorium reilianum) of Sorghum bicolor

The head smut fungus, Sporisorium reilianum ([Kuhn] Landon and Fullerton), was shown to reduce plant height in infected Sorghum bicolor ([L.] Moench) plants. The major reductions occurred in the internodes nearest the panicle and were more severe in naturally infected than in inoculated plants. Less affected plants developed reproductively sterile panicles, and eventually smutted panicles developed phyllodied growths which progressed into leafy shoots. Extracts of smutted, sterile, and healthy (control) panicles of field-grown plants exhibited gibberellin (GA)-like activity in the dwarf rice bioassay. When extracts were purified and assayed with deuterium-labeled GA standards by gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM), GA(1), GA(3), GA(19), GA(20), and GA(53) were detected based on coelution with the standards, identical Kovats retention index values, and matching ion masses and relative abundances for three major ions. In addition, based on published Kovats retention index values, ion masses, and relative abundance values, GA(4), GA(7), GA(8), GA(14), GA(29), and GA(44) were tentatively identified. Quantitative analysis revealed that panicles of healthy control plants contained from 60 to 100% higher total concentrations of GAs than panicles of smutted plants. These comparisons were most striking for the early 13-hydroxylation pathway precursors GA(53), GA(44), and GA(19) but not for GA(20). Extracts of S. reilianum sporidia and culture medium exhibited GA-like bioactivity, and GA(1) and GA(3) were detected based on GC-MS-SIM assay with (2)H-labeled internal standards. Quantitative analysis of these GAs showed increasing concentrations from 4 to 7 to 10 days of culture and a decline at 20 days. This is the first GC-MS-SIM detection of GAs in a non-Ascomycete fungus, and the disease symptoms and quantitative data suggested that fungal infection may interfere with biosynthesis of GAs by the host plant.

Changes after Decapitation in Concentrations of Indole-3-Acetic Acid and Abscisic Acid in the Larger Axillary Bud of Phaseolus vulgaris L. cv Tender Green

Early changes in the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) were investigated in the larger axillary bud of 2-week-old Phaseolus vulgaris L. cv Tender Green seedlings after removal of the dominant apical bud. Concentrations of these two hormones were measured at 4, 6, 8, 12 and 24 hours following decapitation of the apical bud and its subtending shoot. Quantitations were accomplished using either gas chromatography-mass spectrometry-selected ion monitoring (GS-MS-SIM) with [(13)C(6)]-IAA or [(2)H(6)]-ABA as quantitative internal standards, or by an indirect enzyme-linked immunosorbent assay, validated by GC-MS-SIM. Within 4 hours after decapitation the IAA concentration in the axillary bud had increased fivefold, remaining relatively constant thereafter. The concentration of ABA in axillary buds of decapitated plants was 30 to 70% lower than for buds of intact plants from 4 to 24 hours following decapitation. Fresh weight of buds on decapitated plants had increased by 8 hours after decapitation and this increase was even more prominent by 24 hours. Anatomical assessment of the larger axillary buds at 0, 8, and 24 hours following decapitation showed that most of the growth was due to cell expansion, especially in the intermodal region. Thus, IAA concentration in the axillary bud increases appreciably within a very few hours of decapitation. Coincidental with the rise in IAA concentration is a modest, but significant reduction in ABA concentration in these axillary buds after decapitation.

Reversible reaction via a carbanion intermediate in the elimination of ammonia from l-histidine catalysed by histidine ammonia-lyase

l-[5′- 2H 2]Histidine was used as a substrate to investigate the enzymatic reaction mechanism with histidine ammonia-lyase from Pseudomonas fluorescens. The study was performed to determine the exchange rate of deuterium at C-5′ of the imidazole ring with solvent hydrogen relative to the net urocanic acid production. The extent of hydrogen exchange at C-5′ of histidine or urocanic acid was measured by gas chromatography—mass spectrometry—selected ion monitoring, monitoring the molecular ion intensities of the respective gas chromatographic derivatives, at m/z 380 and 379 for histidine and at m/z 267 and 266 for urocanic acid. The observed hydrogen exchange at C-5′ suggested a reversible mechanism via a carbanion intermediate in the reaction with histidine ammonia-lyase.

Cytokinins in the Phloem Sap of White Lupin (Lupinus albus L.)

Cytokinin-like activity in samples of xylem and phloem sap collected from field-grown plants of white lupin (Lupinus albus L.) over a period of 9 to 24 weeks after sowing was measured using the soybean hypocotyl callus bioassay following paper chromatographic separation. The phloem sap was collected from shallow incisions made at the base of the stem, the base of the inflorescence (e.g. stem top), the petioles, and the base and tip of the fruit. Xylem sap was collected as root exudate from the stump of plants severed a few centimeters above ground level. Concentration of cytokinin-like substances was highest in phloem sap collected from the base of the inflorescence and showed an increase over the entire sampling period (from week 10 [61 nanogram zeatin equivalents] to week 24 [407 nanogram zeatin equivalents]). Concentrations in the xylem sap and in the other phloem saps were generally lower. Relatively high concentrations of cytokinin-like substances in petiole phloem sap (70 to 130 nanogram zeatin equivalents per milliliter) coincided in time with high concentrations in sap from the base of the inflorescence (see above). Concentrations in sap (phloem or xylem) from the base of the stem were very much lower. This finding is consistent with movement of cytokinins from leaves into the developing inflorescence and fruit, rather than direct input to the fruit from xylem sap. However, an earlier movement of cytokinins from roots into leaves via the xylem cannot be ruled out. Sap collected at an 18-week harvest was additionally separated by sequential C(18) reversed-phase high performance liquid chromatography --> NH(2) normal phase high performance liquid chromatography, bioassayed, and then analyzed by electron impact gas chromatography-mass spectrometry. Identification of zeatin riboside and dihydrozeatin as two of the major cytokinins in combined sap samples was accomplished by gas chromatography-mass spectrometry-selected ion monitoring.

A selected ion monitoring method for quantifying simvastatin and its acid form in human plasma, using the ferroceneboronate derivative

Simvastatin, a pro-drug lactone, forms the open carboxylic acid as a major metabolite that inhibits the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Simvastatin and the acid in plasma were quantified by a gas chromatography/mass spectrometry/selected ion monitoring (GC/MS/SIM) method. These drugs were separated by solid-phase extraction and independently converted into a 1,3-diol-type compound. This compound reacted with ferroceneboronic acid to yield the cyclic boronate that gave satisfactory mass spectra for GC/MS/SIM measurements. The serum was dominated by the molecular ion appearing as the base peak, thereby leading to a sensitive and selective assay. The calibration curves for simvastatin and the acid were linear in their concentration range of 0.1-10 ng ml-1, where the values of coefficient of variation for both drugs were below 8%, except for the value of 11% for simvastatin at a concentration of 0.1 ng ml-1. The quantification limit for both drugs was 0.1 ng ml-1 on the basis of a signal-to-noise ratio of 4:1.

Dormancy in Peach (Prunus persica L.) Flower Buds

Flower buds of peach (Prunus persica L.) trees, cv Novedad de Cordoba (Argentina), were collected near the end of the dormant period and immediately before anthesis. After removal of scale leaves, morphological observations of representative buds, made on transverse and longitudinal microtome sections, showed that all verticils making up the flower are present in an undifferentiated form during the dormant period (June). Flower buds collected at the end of dormant period (August) showed additional growth and differentiation, at which time formation of two ovules was beginning in the unicarpelar gynoecium. Dehiscence of anthers had not yet occurred 10 days before full bloom, and the ovules were still developing. Free endogenous gibberellin (GA)-like substances were quantified by bioassay (Tan-ginbozu dwarf rice microdrop) after SiO(2) partition column chromatography, reversed phase C18-high performance liquid chromatography, and finally Nucleosil [N(CH(3))(2)]high performance liquid chromatography. Bioactive fractions were then subjected to capillary gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM). Gibberellins A(1), A(3), and A(8) were tentatively identified in peach flower buds using GC-SIM and Kovat's retention indices, and relative amounts approximated by GC-SIM (2:8:6 for GA(1), GA(3), and GA(8), respectively). The highest concentration (330 nanograms per gram dry weight) of free GA(1)/GA(3) was found in dormant buds (June) and diminished thereafter. The concentration free of GA(1)/GA(3) did not increase immediately prior to bud break. However, high GA(1)/GA(3) concentrations occurred during stages where rate of growth and cellular differentiation of (mainly fertile) verticils can be influenced.

Stable Isotope Coadministration Methodology for the Estimation of the Fraction of Imipramine Metabolized to Desipramine

The application of a stable isotope coadministration technique for estimating the fraction (fm) of imipramine (IP) that is converted to desipramine (DMI) is described. Four healthy male subjects received 25mg of IP-d4 hydrochloride orally with 25mg of DMI hydrochloride. The plasma concentrations of IP-d4, DMI-d4, and DMI were determined by capillary gas chromatography-mass spectrometry-selected ion monitoring using d8 analogues as internal standards. The fm values, calculated from the ratio of the area under the plasma concentration–time curve of DMI-d4 to that of DMI, varied from 0.54 to 0.85.

Immunoaffinity Techniques Applied to the Purification of Gibberellins from Plant Extracts

The use of immunoaffinity columns containing anti-gibberellin (GA) antibodies for the selective purification of GAs in plant extracts is described. GA(1), GA(3), GA(4), GA(5), GA(7), and GA(9) conjugates to bovine serum albumin were synthesized and used to elicit anti-GA polyclonal antibodies (Abs) in rabbits. Protein A purified rabbit serum, containing a mixture of anti-GA Abs, was immobilized on matrices of Affi-gel 10 or Fast-Flow Sepharose 4B. Columns of these immunosorbents retained a wide range of C-19 GA methyl esters, but no C-20 GA methyl esters. Quantitative recovery of C-19 GA methyl esters was achieved from the columns, which, after reequilibration in buffer, could be reused up to 500 times. The immunosorbents were tested by examination of extracts from immature soybean and pea seeds. GAs were initially purified by passing the extracts through DEAE-cellulose and concentrating them on octadecylsilica. The extracts were methylated and further purified on the mixed anti-GA immunoaffinity columns. GAs were detected and quantified as methyl esters or methyl ester trimethylsilyl ethers by gas chromatography-mass spectrometry-selected ion monitoring. GA(7) was found in soybean seeds, 17 days after anthesis, at low levels (8.8 nanograms per gram fresh weight). C-19 GAs were examined in cotyledons, embryonic axes, and testae of G2 pea seeds harvested 20 days after anthesis. High levels of GA(20) and GA(29) were found in cotyledons (3580 and 310 nanograms per gram fresh weight, respectively) and embryonic axes (5375 and 1430 nanograms per gram) fresh weight, respectively). Lower levels of GA(9) were found in cotyledons and embryonic axes (147 and 161 nanograms per gram fresh weight, respectively). GA(9) was the major GA of testae at levels of 195 nanograms per gram fresh weight. Trace quantities of GA(20) and GA(51) were also observed in testae.

Lithocholate in liver tissue with obstructive jaundice.

The existence of tissue-bound lithocholate in liver tissue was first suggested by Nair et al. (Lipids 1988;12:922-929). Since lithocholate is well known to be cholestatic, we investigated whether tissue-bound lithocholate, if present, can be correlated to liver dysfunction in obstructive jaundice. Tissue-bound lithocholate in liver tissues of 7 patients with obstructive jaundice was analyzed using gas chromatography-mass spectrometry-selected ion monitoring. We found no accumulation of lithocholate, including tissue-bound form, indicating that the role of lithocholate in inducing hepatic dysfunction in obstructive jaundice is minimal, if any.

A Gibberellin-Deficient Brassica Mutant—rosette

A single-gene mutant (rosette [ros/ros]) in which shoot growth and development are inhibited was identified from a rapid cycling line of Brassica rapa (syn campestris). Relative to normal plants, the mutant germinated slowly, had delayed or incomplete floral development, and reduced leaf, petiole, and internode growth. The exogenous application of GA(3) by foliar spray or directly to the shoot tip of rosette resulted in rapid flowering, bolting (shoot elongation), and viable seed production. Shoots of rosette contained endogenous levels of total gibberellin (GA)-like substances (;Tan-ginbozu' dwarf rice assay) of about one-tenth of that of the normal rapid-cycling line of B. rapa which consisted almost entirely of a very nonpolar, GA-like substance which yielded GA(1) and GA(3) upon mild acid hydrolysis. In a normal rapid-cycling B. rapa line, the nonpolar putative GA(1) and GA(3) conjugates were present, but additionally, free GA(1) and GA(3) were abundant and identified by gas chromatography-mass spectrometry-selected ion monitoring. The quantities of free GA(1) and GA(3) in the normal line and in rosette were quantified by GC-MS-SIM using [(2)H(2)]GA(1) as an internal standard. Fourteen-day-old rosette and normal seedlings contained 5.3 and 23.2 ng GA(1) per plant, respectively. At day 21 the rosette plants contained 7.7 and 26.1 nanograms per plant of GA(1) and GA(3), while normal plants contained 31.1 and 251.5 nanograms per plant, respectively. Thus, normal plants contained from four to ten times higher levels of total GA-like substances, GA(1), or GA(3), than rosette. The ros allele results in reduced GA level, yielding the rosette phenotype whose delayed germination and flowering, and reduced shoot growth responses indicate a probable role for endogenous GA(1) and GA(3) in the regulation of these processes in Brassica.

Endogenous and exogenous testosterone levels after administration of deuterium-labelled testosterone propionate in hypogonadotropic hypogonadism.

A gas chromatography-mass spectrometry-selected ion monitoring technique was employed to determine simultaneously the plasma concentrations of endogenous and exogenous testosterone in three patients with hypogonadotropic hypogonadism after a single i.m. dose of testosterone propionate-19,19,19-d3. The plasma levels of testosterone-19,19,19-d3 derived from testosterone propionate-19,19,19-d3 were maintained above the normal testosterone levels (greater than 4 ng/ml) for 48 h, while the plasma levels of endogenous testosterone changed little.

Selected ion monitoring analysis of NB-818, a new calcium entry blocker in a series of dihydropyridines, in human plasma.

NB-818 is expected in clinical use to produce an antihypertensive effect and simultaneously to promote an increase in cerebral blood flow, owing to its vasodilator activity. To determine NB-818 levels in human plasma for pharmacokinetic studies, a capillary column gas chromatography/mass spectrometry/selected ion monitoring (GC/MS/SIM) method was employed. Because NB-818 was degraded under GC conditions, stable derivatives had to be prepared for GC/MS measurements. Reaction of NB-818 with silylation agents produced derivatives quantitatively. In the silylation, however, the carbomoyl group of NB-818 was replaced by silyl moieties. NB-818 is possibly metabolized at the carbamate moiety alone, giving a compound with a hydroxymethyl group in place of the carbamoyloxymethyl group. Since this compound and NB-818 gave the same derivative, efforts were devoted to separating them. A detection limit below 0.1 ng ml-1 was attained. The calibration curve was linear in the concentration range 0.1-10 ng ml-1 (r = 0.9998), and the coefficient of variation was 5.5% and 3.1% at concentrations of 1 and 10 ng ml-1, respectively.

Determination of Abscisic Acid in Pinus densiflora by Selected Ion Monitoring

Abscisic acid (ABA) in the stem of akamatsu (Pinus densiflora) was identified and quantified by gas chromatography-mass spectrometry-selected ion monitoring using hexadeuterated ABA as an internal standard. tert-Butyldimethylsilyl ester was used as a derivative of ABA. This derivative had high sensitivity and selectivity for ABA determination. ABA concentrations in cambial region scrapings were independent of the cessation of cambial activity.

Ultraviolet-induced accumulation of phytoalexins in rice leaves.

Accumulated phytoalexins in ultraviloet-irradiated detached rice leaves were detected by gas chromatography-mass spectrometry-selected ion monitoring (GC-MS-SIM) after pretreating with a BOND ELUT C18 cartridge. An accumulation of oryzalexins A, B, C and D, momilactones A and B, and an unknown antifungal substance accompanied by the appearance of brown spots on the leaf surface was observed. Momilactone A was detected in abundance, and among the oryzalexins, oryzalexin D was a major substance. The maximum accumulation of these phytoalexins, except for oryzalxins C and D, was found 3 days after irradiation. However, the maximum accumulation of oryzalexin D was observed 2 days after irradiation and that of oryzalexin C was observed 4 days after it. The content of these diterpenoid phytoalexins in rice leaves was dependent on leaf aging, an accumulation of these phytoalexins in the uppermost leaves being much lower than that in the aged leaves (lower leaves), and brown spots scarcely ever appeared on the sur...

Selected-ion monitoring gas chromatographic—mass spectrometric analysis of catecholamines: enhancement of sensitivity by a simple clean-up step on Sephadex G-10

The sensitivity of catecholamines using selected-ion monitoring gas chromatography—mass spectrometry (SIM GC—MS) has been enhanced by employing a simple clean-up step using Sephadex G-10 columns. This procedure allows for the detection of extremely small amounts (1–10 pg) of biogenic amines (e.g., epinine) which corresponds to a 25-fold increase in detection limits compared to electron ionization GC—MS or high-performance liquid chromatography with electrochemical detection (HPLC—ED). The SIM GC—MS assay was used to monitor the dopamine-β-hydroxylase-mediated conversion of epinine d6 to epinephrine d6 in rat hypothalamus and brainstem in vitro and the results were compared to those obtained by HPLC—ED.

Identification and quantitation of trenbolone in bovine tissue by gas chromatography-mass spectrometry

Identification and quantitation of trace amounts of trenbolone in bovine tissue by capillary gas chromatography-mass spectrometry-selected-ion monitoring (GC-MS-SIM) has been developed. Three-phase liquid-liquid extraction using a mixture of water-acetonitrile-dichloromethane-hexane was utilized for the sample extraction from tissue. Target compounds were extracted from the tissue into the acetonitrile layer. The residue from this extraction was then subjected to solid-phase extraction by C 18 and silica gel disposable cartridges using methanol-water and benzene-acetone as eluents. To overcome extensive matrix interferences, preparative reversed-phase high-performance liquid chromatographic separation was used with an octadecyl-bonded column using methanol-water as mobile phase for sample clean-up prior to GC-MS analysis. A structural analogue of trenbolone, 19-nortestosterone, was chosen as the internal standard for quantitation by GC-MS. The sample was co-injected with N,O-bis(trimethylsilyl)trifluoroacetamide-1-(trimethylsilyl) imidazole (95:5, v/v) for flash heater derivatization. Identification and quantitation were simultaneously carried out by SIM of characteristic ions of the trimethylsilyl derivatives of trenbolone and 19-nortestosterone. The limit of detection for trenbolone and epitrenbolone was 0.5 ppb in muscle and liver tissue. A comparison of sensitivity and specificity between GC-MS under electron ionization in addition to positive- and negative-ion chemical ionization conditions using methane reagent gas is also discussed.

Measurement of Indole-3-Acetic Acid in Peach Fruits (Prunus persica L. Batsch cv Redhaven) during Development

The amount of indole-3-acetic acid (IAA) was measured in peach fruits by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution assay with [(13)C(6)]IAA as an internal standard throughout the growing season. Ethylene evolution of the fruit was also measured. IAA levels were 25 nanograms per gram fresh weight, 18 days after anthesis. Both IAA levels and rates of ethylene evolution declined to their lowest levels (7 nanograms IAA per gram fresh weight and 0.01 nanoliter ethylene per gram per hour) in the second stage of fruit growth. Endogenous levels of free-IAA and ethylene evolution increased in the last stage of peach fruit development to 32 nanograms per gram fresh weight and 0.27 nanoliter per gram per hour, respectively. IAA amounts peaked in the ovules 67 days after anthesis.

QSIMPS: A response to the analytical challenge of the pharmacokinetic revolution

Pharmacokinetics is a collection of equations (and concepts) which can transform blood and/or urine concentration—time data for a drug into numerical parameters that are useful as practical instruments for deciding certain questions concerning the efficacy and safety of the drug. To generate the parameters requires the analysis of hundreds of samples; experiments to compare parameters require the analysis of thousands of samples. Typically, tens of thousands of analyses are needed to generate the pharmacokinetic data required to bring a new drug on the market. This paper describes QSIMPS, a collection of software and hardware for the rapid, automated collection, processing and analysis of gas chromatography—mass spectrometry—selected ion monitoring data acquired for use in pharmacokinetic studies.

Identification of Endogenous Gibberellins in the Winter Annual Weed Thlaspi arvense L

Eleven endogenous gibberellins (GAs) were identified by combined gas chromatography-mass spectrometry in purified extracts from shoots of field pennycress (Thlaspi arvense L.): GA(1,9,12,15,19,20,24,29,44,51,53). Traces of GA(8) and GA(25) were tentatively indicated by combined gas chromatography-mass spectrometry-selected ion monitoring. Comparison of the total ion current traces indicated that GA(19) and GA(44) were most abundant, while GA(12,15,20,24,29,53) occurred in lesser amounts. Only small amounts of GA(1,9,51) were present. The levels of GA(8) and GA(25) were barely detectable. Consideration of hydroxylation patterns of the ent-gibberellane ring structure indicates two families of GAs: one with a C-13 hydroxyl group (GA(1,8,19,20,29,44,53)) and another whose members are either nonhydroxylated (GA(9,12,15,24,25)) or lack a C-13 hydroxyl group (GA(51)). This suggests that in field pennycress there are two parallel pathways for GA metabolism with an early branch point from GA(12): an early C-13 hydroxylation pathway, leading ultimately to GA(1) and GA(8) and a C-13 deoxy pathway culminating in the formation of GA(9) and GA(51).

Selective gas chromatographic analysis of chlorinated phenolic compounds using the nitrogen—phosphorus detector

A new gas chromatographic method, using a nitrogen—phosphorous detector to selectively analyse chlorinated phonelic compounds, is described and its usefulness evaluated. In this scheme of analysis the chlorinated phenolic substances are converted into their cyanoethyldimethylsilyl ether derivatives by a one-step reaction with 2-cyanoethyldimethyl(diethyl)aminosilane prior to analysis. Results indicate that the ethers have good gas chromatographic properties and can be detected with high sensitivity by the nitrogen—phosphorus detector. The response is linear over a wide range of concentrations and the limits of detection are (5 +− 2) · 10 −3 g N. Furthermore, the relative response factors calculated for chlorophenols and chlorinated hydroxybiphenyls. when using this detector, are close to unity. The mass spectrometric behavior of cyanoethyldimethylsilyl ethers is also discussed with reference to structure specificity and detection at low levels by selected-ion monitoring gas chromatography—mass spectrometry.