Chromatography-selected Ion Monitoring-mass Research Articles

Multi-Residual Determination of Multi-Class Pesticides in Groundwater by Direct Immersion Solid-Phase Microextraction with Gas Chromatography-Selected Ion Monitoring Mass Spectrometry (GC–MS/SIM) Detection

Multi-Residual Determination of Multi-Class Pesticides in Groundwater by Direct Immersion Solid-Phase Microextraction with Gas Chromatography-Selected Ion Monitoring Mass Spectrometry (GC–MS/SIM) Detection

Development and Validation of a HS-SPME-GC-SIM-MS Multi-Method Targeting Hop-Derived Esters in Beer

Hop-derived esters contribute to beer flavor and changes in the ester spectrum are believed to be an important driver in the flavor–instability of hoppy beers. To date, there exists no published method that enables reliable quantification of hop-derived esters in beer. As the availability of such a method is vital to enlarge the understanding of beer flavor stability, the current article is concerned with the development, validation, and application of a headspace solid phase microextraction gas chromatography selected ion monitoring mass spectrometry (HS-SPME-GC-SIM-MS) based methodology for quantification of 16 hop-derived esters in beer. The validation data shows that choosing suitable HS-SPME-GC-SIM-MS conditions enabled reliable quantification of esters across a range of 1–200 µg/L, with calculated limits of quantification being well below 1 µg/L. Spiking experiments using terpenes and terpenoids evidenced method robustness, most importantly when two commercially available stable isotope labeled internal standards (d6-geranyl acetate and 13C-methyl octanoate) were used. Application to beer samples indicated that beers brewed with different qualities/quantities of hops and differing in freshness could be well differentiated by HS-SPME-GC-SIM-MS analysis of their ester profile. Supplemental data for this article is available online at at https://doi.org/10.1080/03610470.2021.1994814 .

Determination of 24 Pesticides Residues in Leather Products by Solid-Phase Microextraction Coupled with Gas Chromatography–Mass Spectrometry

Considering the high content of oil and complex residual additives in leather samples, a new analytical method based on the solid-phase extraction technique and gas chromatography-selected ion monitoring mass spectrometry (GC-SIM-MS) was developed to determine 24 organic compounds involving the organochlorine pesticides (OCPs), organophosphorous pesticides (OPPs) and pyrethroids pesticides residues in leather. The extraction conditions (such as the extraction solution, purification procedure and solid-phase extraction column) were optimized using the positive leather samples based on the recovery rates of the pesticides. The best extraction solution, solid-phase extraction column and chromatography column were n-hexane and ethyl acetate (1+1, volume) mixed solution, Carb-PSA (1.0 g, 6mL) and DB-1701 (length: 30 m, inside diameter: 0.25 mm, film thickness: 0.25 μm). The optimized extraction time and temperature were 20 min and 25°C, respectively. The detection limits of 24 pesticide residues range from 0.05 to 0.10 mg/kg, and the recoveries range from 74% to 116%. The relative standard deviations (RSD, n=6) range from 5.42% to 12.00%. The developed method presented a simple, rapid, sensitive, and inexpensive method to detect 24 pesticides in skin and leather and was successfully applied to the detect them in leather products (cowhide, sheep leather and pig leather).

A Selective Gas Chromatography-Selected Ion Monitoring Mass Spectrometry (GC‑MS/SIM) Method to Determine Polychlorinated Biphenyls in Fuller's Earth

Polychlorinated biphenyls (PCBs) are carcinogenic to humans and can be found in Fuller’s earth used for the regeneration of used transformer oil. This work describes a highly selective gas chromatographic-selected ion monitoring mass spectrometric method (GC-MS/SIM) for the simultaneous determination of three Aroclor PCB congener mixtures (1242, 1254, and 1260). The separation of the PCBs could be systematically varied by changing the temperature conditions of the chromatographic column. Methods were first selected from the literature, and the resolution of the PCBs was then optimized by adjusting k* (effective retention), a (selectivity), and N (column efficiency), enabling the detection of 83 PCBs in 35 min. The extract of PCBs from Fuller’s earth was obtained by ultrasonication (employing hexane and acetone as solvents) and cleanup with Florisil and was concentrated to about 500 µL. The ions analyzed in SIM mode were divided into groups according to the similarity (m/z) of the PCB fragmentation profiles. The limits of detection (LOD) and quantification (LOQ) were 0.19 and 0.64 µg kg-1, respectively. The method developed was used to determine the PCBs present in Fuller’s earth provided by an electric power company. The mean total PCBs concentration was found 1.9 µg kg-1 in Fuller’s earth.

Exploring metabolic syndrome serum free fatty acid profiles based on GC–SIM–MS combined with random forests and canonical correlation analysis

Metabolic syndrome (MetS) is a cluster of metabolic abnormalities associated with an increased risk of developing cardiovascular diseases or type II diabetes. Till now, the etiology of MetS is complex and still unknown. Metabolic profiling is a powerful tool for exploring metabolic perturbations and potential biomarkers, thus may shed light on the pathophysiological mechanism of diseases. In this study, fatty acid profiling was employed to exploit the metabolic disturbances and discover potential biomarkers of MetS. Fatty acid profiles of serum samples from metabolic syndrome patients and healthy controls were first analyzed by gas chromatography–selected ion monitoring–mass spectrometry (GC–SIM–MS), a robust method for quantitation of fatty acids. Then, the supervised multivariate statistical method of random forests (RF) was used to establish a classification and prediction model for MetS, which could assist the diagnosis of MetS. Furthermore, canonical correlation analysis (CCA) was employed to investigate the relationships between free fatty acids (FFAs) and clinical parameters. As a result, several FFAs, including C16:1n-9c, C20:1n-9c and C22:4n-6c, were identified as potential biomarkers of MetS. The results also indicated that high density lipoprotein-cholesterol (HDL-C), triglycerides (TG) and fasting blood glucose (FBG) were the most important parameters which were closely correlated with FFAs disturbances of MetS, thus they should be paid more attention in clinical practice for monitoring FFAs disturbances of MetS than waist circumference (WC) and systolic blood pressure/diastolic blood pressure (SBP/DBP). The results have demonstrated that metabolic profiling by GC–SIM–MS combined with RF and CCA may be a useful tool for discovering the perturbations of serum FFAs and possible biomarkers for MetS.

Authentication of pineapple (Ananas comosus [L.] Merr.) fruit maturity stages by quantitative analysis of γ- and δ-lactones using headspace solid-phase microextraction and chirospecific gas chromatography–selected ion monitoring mass spectrometry (HS-SPME–GC–SIM-MS)

Headspace solid phase microextraction and chirospecific gas chromatography–mass spectrometry in selected ion monitoring mode (HS-SPME–GC–SIM-MS) allowed quantitative determination of δ-lactones (δ-C8, δ-C10) and γ-lactones (γ-C6, γ-C8, γ-C10). A stable isotope dilution assay (SIDA) with d7-γ-decalactone as internal standard was used for quantitative analysis of pineapple lactones that was performed at three progressing post-harvest stages of fully ripe air-freighted and green-ripe sea-freighted fruits, covering the relevant shelf-life of the fruits. Fresh pineapples harvested at full maturity were characterised by γ-C6 of high enantiomeric purity remaining stable during the whole post-harvest period. In contrast, the enantiomeric purity of γ-C6 significantly decreased during post-harvest storage of sea-freighted pineapples. The biogenetical background and the potential of chirospecific analysis of lactones for authentication and quality evaluation of fresh pineapple fruits are discussed.

Determination of Polycyclic Aromatic Hydrocarbons in Coffee and Coffee Substitutes using Dispersive SPE and Gas Chromatography-Mass Spectrometry

The purpose of this study was to evaluate the application of dispersive solid-phase extraction with the use of primary secondary amine (PSA), NH2 and strong anion exchange (SAX) sorbents for polycyclic aromatic hydrocarbon (PAH) determination in samples of coffee. The final extracts were analysed by gas chromatography selected ion monitoring mass spectroscopy (GC-SIM-MS). The results indicated that cleanup with PSA and SAX sorbents followed by final solvent exchange from acetonitrile to hexane (liquid–liquid extraction, LLE) was the best variant of the method, resulting in the cleanest extracts and satisfactory compound recoveries (70–104 %) for examined compounds in coffee samples. In the study of real samples, the concentrations of PAH markers (benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[a]pyrene) were higher for natural roasted coffee (7.20–68.15 μg kg−1), than for instant coffee (2.97–19.55 μg kg−1) and cereal coffee (8.15–15.35 μg kg−1), and the sum of PAHs was also higher in natural and instant coffees compared to coffee substitutes (364, 343 and 265 μg kg−1, respectively). However, in all samples of coffee, light (three-ring) PAHs dominated within the identified compounds and phenanthrene was the most abundant compound, approaching 209.67 μg kg−1 in roasted coffee.

Metal-tag labeling coupled with high performance liquid chromatography-selected ion monitoring mass spectrometry for absolute quantitation of proteins

A novel method has been established based on metal element chelated tags coupled with high performance liquid chromatography-selected ion monitoring mass spectrometry (HPLC-SIM/MS). The labeling efficiency and stability of metal element chelated tags, the chromatographic retention behavior and MS behavior of the labeled peptides, the linear range and accuracy of this method were examined. The results showed that the metal element chelated tag method has high labeling efficiency and high labeling stability, and the labeled peptides with different kinds of metal tags have consistent chromatographic retention behavior. The method of metal tags coupled with HPLC-SIM/MS has high sensitivity with the limit of quantification (LOQ) up to 1 fmol. The linear range for the method was between 1 fmol to 500 fmol with R2 > 0.99, which means the method has a good linearity. Moreover, this method had an average recovery of 117.01%. The method was used in the absolute quantitation of a protein enolase in Thermoanaerobacter tengcongensis (TTE) with a relative standard deviation of 5.74%, which means high precision. All the results showed that this method is accurate and reliable for the absolute quantitation of proteins. This gives us an alternative for the quantitative determination of proteins in relatively simple biological samples.

Cork as a new (green) coating for solid-phase microextraction: Determination of polycyclic aromatic hydrocarbons in water samples by gas chromatography–mass spectrometry

A new fiber for solid-phase microextraction (SPME) was prepared employing cork as a coating. The morphology and composition of the cork fiber was evaluated by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), respectively. The proposed fiber was used for the determination of polycyclic aromatic hydrocarbons (PAHs) in river water samples by gas chromatography–selected ion monitoring–mass spectrometry (GC–SIM–MS). A central composite design was used for optimization of the variables involved in the extraction of PAHs from water samples. The optimal extraction conditions were extraction time and temperature of 60min and 80°C, respectively. The detection and quantification limits were 0.03 and 0.1μgL−1, respectively. The recovery values were between 70.2 and 103.2% and the RSD was ≤15.7 (n=3). The linear range was 0.1–10μgL−1 with r≥0.96 and the fiber-to-fiber reproducibility showed RSD≤18.6% (n=5). The efficiency of the cork fiber was compared with commercially available fibers and good results were achieved, demonstrating the applicability and great potential of cork as a coating for SPME.

Metabolite target analysis of isoprenoid pathway in Saccharomyces cerevisiae in response to genetic modification by GC-SIM-MS coupled with chemometrics

A highly selective, sensitive and nonradioactive analytical method for identification and quantification of intracellular metabolites involved in isoprenoid pathway has been developed by means of gas chromatography-selected ion-monitoring mass spectrometry (GC-SIM-MS). These metabolites are classified into two groups: sterols (squalene, ergosterol, lanosterol) and phosphorylated compounds (geranyl diphosphate, farnesyl pyrophosphate, geranylgeranyl pyrophosphate) based on their physicochemical properties. To quantify both groups in a single analytical run, GPP, FPP and GGPP were cleaved to the parent alcohols, geraniol, farnesol, geranylgeraniol by pyrophosphatase followed by alkaline phosphatase before extraction, separation and detection. This study evaluated several extraction procedures and determined the effects of the type of extraction solvent, times used for extraction. Under optimized GC/EI-MS conditions, six compounds were separated with high efficiency in the selected-ion monitoring (SIM) mode. Linearity of the method was good with correlation coefficients (r 2) in the range of 0.9953–0.9999 and detection limits were 1.53–151.88 ng/ml. The intra-day and inter-day precision of the method, as RSD, were less than 5.31 and 6.04%, respectively. The accuracy of six compounds varied between 87.7 and 110.8%. This assay was successfully applied to the determination of six major metabolites in the pathway for isoprenoid biosynthesis in S. cerevisiae and is sensitive to detect changes following genetic modification. By isolating statistically significant differences among metabolite levels from four biological conditions, we observed discriminatory metabolic features that hinted that the role of erg9 and coq1gene was involved in isoprenoid pathway. Integrating this analytical approach with statistical strategies, we can determine the influence of erg9 and coq1gene on isoprenoid levels of S. cerevisiae, thus leading to improved understanding of the pathway in a multitude of biological systems.

A capillary gas chromatography-selected ion monitoring mass spectrometry method for the analysis of atractylenolide I in rat plasma and tissues, and application in a pharmacokinetic study

The aim of this paper is to investigate the characteristics of atractylenolide I (AO-I) in the body by a GC–MS method. All bio-samples were cleared up with a liquid–liquid extraction procedure. The calibration curves were linear within a range of 5–1000 ng/mL for plasma samples, 0.06–16.00 μg/g for cerebellum samples, and 0.03–8.00 μg/g for other tissue samples. The limit of quantification (LOQ) for AO-I was 1.0 ng/mL or 1.0 ng/g (S/N ≥ 10) in the bio-samples. In the applications, the main pharmacokinetic parameters were firstly obtained as follows: T max = 0.37 ± 0.19 h, C max = 0.26 ± 0.05 μg/mL, AUC = 1.95 ± 0.30 μg h/mL and k a = 10.08 ± 5.60 h −1. The tissue distribution of AO-I in rats after the oral administration of 50.0 mg/kg was from 0.225 to 0.031 μg/g with a decreasing tendency in different tissues like liver > kidney > spleen > cerebellum > heart > cerebrum > lung. The protein binding in rat plasma, human plasma and bovine serum albumin was 80.8 ± 3.9, 90.6 ± 3.1 and 60.9 ± 5.1%, respectively.

Validated semiquantitative/quantitative screening of 51 drugs in whole blood as silylated derivatives by gas chromatography-selected ion monitoring mass spectrometry and gas chromatography electron capture detection

A comprehensively validated procedure is presented for simultaneous semiquantitative/quantitative screening of 51 drugs of abuse or drugs potentially hazardous for traffic safety in serum, plasma or whole blood. Benzodiazepines (12), cannabinoids (3), opioids (8), cocaine, antidepressants (13), antipsychotics (5) and antiepileptics (2) as well as zolpidem, zaleplon, zopiclone, meprobamate, carisoprodol, tizanidine and orphenadrine and internal standard flurazepam, were isolated by high-yield liquid-liquid extraction (LLE). The dried extracts were derivatized by two-step silylation and analyzed by the combination of two different gas chromatographic (GC) separations with both electron capture detection (ECD) and mass spectrometry (MS) operating in a selected ion-monitoring (SIM) mode. Quantitative or semiquantitative results were obtained for each substance based on four-point calibration. In the validation tests, accuracy, reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, as well as extraction efficiency and stability of standard stock solutions were tested, and derivatization was optimized in detail. Intra- and inter-day precisions were within 2.5–21.8 and 6.0–22.5%, and square of correlation coefficients of linearity ranged from 0.9896 to 0.9999. The limit of quantitation (LOQ) varied from 2 to 2000 ng/ml due to a variety of the relevant concentrations of the analyzed substances in blood. The method is feasible for highly sensitive, reliable and possibly routinely performed clinical and forensic toxicological analyses.

Determination of corticosterone in rat and mouse plasma by gas chromatography-selected ion monitoring mass spectrometry

A simple, highly sensitive and specific method based on gas-chromatography-selected ion monitoring (SIM) mass spectrometry has been developed for the quantitation of corticosterone in rat and mouse plasma. After extraction of the plasma with ethyl acetate, the residue was trimethy-silylated with pentafluorobenzyl hydroxylamine-trimethylsilyl (PFBO-TMS). Detection of the derivatives was accomplished by a quadruple mass spectrometer in the selected ion monitoring mode ( m/ z of 316, 648, 663 and 678). The detection limit of the assay was 0.1 pg on column. The results show that in the plasma of non-stressed animals, only minor amounts of corticosterone were found; whereas in the plasma of stressed animals, it was dramatically increased. The method developed here can be used to examine corticosterone levels as a marker of stress in rats and mice and may also be used for estimation of the effect of stress-release medications.

Sequences of polypeptide antibiotics stilboflavins, natural peptaibol libraries of the mold Stilbella flavipes.

From the culture broths of the mold Stilbella flavipes CBS 146.81, a mixture of polypeptides could be isolated by adsorption on XAD polystyrene resin and purified by Sephadex LH-20 chromatography. Using preparative thin-layer chromatography (TLC) three groups of peptides, named stilboflavins (SF) A, B, and C could be separated. Each of the groups showed microheterogeneity when investigated by high-performance liquid chromatography (HPLC). Employing on-line HPLC-electrospray ionization tandem mass spectrometry in the positive and negative ionization mode, together with gas chromatography-selected ion monitoring mass spectrometry, enantioselective GC and quantitative amino acid analysis, the sequences of stilboflavins A and B could be determined. Exchange of Glu in stilboflavins A peptides (acidic) against Gln in stilboflavins B peptides (neutral) is the rational for different polarity of the peptide groups and their separatability by TLC. Since SF A and B are bioactive N-acetylated 20-residue peptides with a high proportion of alpha-aminoisobutyric acid and C-terminal bonded amino alcohols (either leucinol, isoleucinol or valinol) the peptides belong to the group of peptaibol antibiotics.

Determination of testosterone and 6β-hydroxytestosterone by gas chromatography–selected ion monitoring–mass spectrometry for the characterization of cytochrome p450 3A activity

A method for the determination of testosterone and its metabolite, 6β-hydroxytestosterone, in liver microsomal incubates employing gas chromatography with selected ion monitoring mass spectrometric detection (GC–SIM–MS) has been developed. The method is more rapid than previously reported methods. Testosterone and its metabolites are extracted from the incubation mixture in a single step with methylene chloride. The method does not require derivatization and testosterone and its metabolites are separated on a HP-5MS fused-silica capillary column in less than 15 min. The retention times of testosterone ( m/z 288), methyltestosterone ( m/z 302), and 6β-hydroxytestosterone ( m/z 304) are approximately 12.7, 12.8, and 13.4 min, respectively. There are no interferences from other known CYP450 metabolites of testosterone. In addition, the selectivity and specificity of the mass spectrometer helps eliminate possible interferences from drugs and new chemical entities evaluated using this methodology. Calibration curves for testosterone and 6β-hydroxytestosterone are linear from 0.25 to 100 μ M. Extraction recoveries are better than 92% for both analytes and the internal standard, methyltestosterone. Over the course of five separate runs, within-day and inter-day precision (expressed as relative standard deviation) was less than 5% for all concentrations of testosterone and 6β-hydroxytestosterone. Accuracies ranged from 95.8 to 105.8% for testosterone and 94.6 to 104.2% for 6β-hydroxytestosterone. The assay has been used to characterize the CYP3A metabolic activity of multiple preparations of human, rat, and dog liver microsomes.

Determination of testosterone in human hair by gas chromatography-selected ion monitoring mass spectrometry.

A method for the determination of testosterone in human hair by gas chromatography-mass spectrometry using d3-testosterone as internal standard is described. Our method consisted of alkaline digestion, fast liquid-liquid extraction, LH-20 chromatography and derivatization with heptafluorobutyric anhydride. Quantification was achieved by selected ion monitoring of m/z 680 (testosterone) and m/z 683 (d3-testosterone). Our method needed no complex corrections for isotope contributions. The procedure provided a sensitive and specific technique with good accuracy and precision. For the first time, testosterone has been quantified by gas chromatography-mass spectrometry in human hair. The concentrations (median, range, ng g-1 hair) reflected a significant (p = 0.05; t-test) sex difference with 2.7 ng g-1 (2.5-4.2) in male and 1.7 ng g-1 (1.0-3.4) in female hair.

Determination of low levels of poly(ethylene glycol) 400 in plasma and urine by capillary gas chromatography-selected ion-monitoring mass spectrometry after solid-phase extraction

A convenient and sensitive method for the quantitative determination of poly(ethylene glycol) 400 in plasma and urine with capillary gas chromatography-mass spectrometry has been developed. The sample preparation involves solid-phase extraction with subsequent derivatization with heptafluorobutyric anhydride, which proved to give the most stable derivative. The derivatization procedure was optimized using experimental design, and different solid-phase extraction columns were evaluated. The limit of quantitation was 1 μmol/l (0.4 μg/ml) for both plasma and urine.

Evaluation of D-amino acid levels in rat by gas chromatography-selected ion monitoring mass spectrometry: no evidence for subacute toxicity of orally fed D-proline and D-aspartic acid.

Sprague-Dawley rats received deionized water (controls) during 28 days or drinking water with added D-proline, L-proline, D-aspartic acid or L-aspartic acid corresponding to a mean daily load of approximately 50 mg amino acid enantiomer kg-1 body weight. Parameters indicating the physiological status (food intake and body weight, glutamic-oxalic-transaminase, glutamic-pyruvic-transaminase, alkaline phosphatase, urea and creatinine in serum, and creatine and osmomolality of urine) were determined. After 28 days the weights of the supposed target organs of toxicity (kidney, liver, brain, thymus) were determined and organs were inspected for macroscopic and microscopic alterations. No pathological changes in the organs were observed and no signs of subacute toxicity (liver, kidney) were found. In serum, homogenates of liver, kidney and brain, and in part, in urine, the amounts of D-amino acids (D-AAs) were quantitatively determined using chiral phase capillary gas chromatography-selected ion monitoring mass spectrometry. Significant levels of certain D-AAs (Ala, Pro, Ser, Asx, Glx, Orn and Lys) were already detectable in kidney and liver homogenates and serum of controls. In brain homogenates the highest amounts among the D-AAs were found for D-Ser (up to 382 nmol g-1), moderate amounts for D-Ala, D-Asx and D-Glx, and, in a few cases, trace amounts for D-Orn and D-Lys (1-2 nmol g-1). D-Pro was not detected either in the brains of controls or in the brains of animals loaded with D-Pro. Feeding with D-Pro resulted in a 20-30 fold increased renal excretion of D-Pro at the end of the experiment. Continuous feeding with D-Asp did not increase renal excretion of this enantiomer, but in the serum, higher amounts (0.8-4.0 mumol-1) were determined in comparison to the control group (0.3-0.9 mumol-1). Feeding with D-Pro led to an increase of this enantiomer in serum (1.3-10.5 mumol-1). Feeding with D-Asp did not increase its amounts in brain homogenates (38 and 43 nmol g-1) in comparison to controls.

Incorporation of Deuterium into Indole-3-acetic Acid and Tryptophan in Zea mays Seedlings Grown on 30 % Deuterium Oxide

Summary The onset of de novo synthesis of IAA and tryptophan in seedlings of Zea mays sweet corn, var. Silver Queen was examined. Deuterium labeled water was used as a general precursor to minimize assumptions as to the biosynthetic route. Protium in positions 2, 4, 5, 6, & 7 of the indole ring are non-exchangeable (Magnus et al., 1980). IAA and tryptophan synthesized via the shikimic acid pathway would contain deuterium in one or more of these positions. Protium on the indene nitrogen, the carboxyl, the amino group (tryptophan only), or the protium alpha to the carboxyl (IAA only), exchange readily and were removed by base catalyzed exchange prior to GGMS analysis. We have demonstrated, by means of gas chromatography-selected ion monitoring mass spectrometry, that 7 d old, etiolated seedlings grown on 30 % D 2 O incorporate deuterium into tryptophan, but not into IAA. Randomization of deuterium in the indole ring during mass spectrometry makes it difficult to localize the deuterium to the tryptophan ring. Deuterium NMR was used for the determination of the position of deuterium incorporated into tryptophan. NMR data are presented showing deuterium incorporation into the 6 position of the tryptophan indole ring.

Ethylene Biosynthesis and Ethephon Metabolism and Transport in Barley

When applied to barley (Hordeum vulgare L.) for lodging control, ethephon (2‐chloroethylphosphonic acid) also promotes tillering. These tillers do not mature prior to harvest, and may compete for assimilates and reduce yield. This combined field and greenhouse study was conducted to investigate the possibility that promotion of tillering by ethephon includes (i) an effect of ethephon on ethylene biosynthesis, (ii) ethephon transport and metabolism, and (iii) a direct effect of ethephon in the plant base near the tiller buds. Ethylene release from flag leaves, spikes, and stems was determined by photoionization gas—liquid‐chromatography, 1‐Aminocyclopropane‐1‐earboxylic acid (ACC) and conjugated ACC were quantified by combined gas chromatography—selected ion monitoring—mass spectroscopy. Ethephon transport was monitored with [14C]ethephon, and by measuring ethylene release from untreated plant parts. Ethephon treatment resulted in a substantial increase in ethylene release from both flag leaves and spikes, peaking 1 to 2 d after treatment and remaining above controls for at least 15 d after treatment. No effect of treatment was observed on ACC and conjugated ACC levels; thus, ethephon application did not alter endogenous ethylene biosynthesis. Decreasing amounts of [14C]ethephon were observed (TLC analysis) in the flag leaves and spikes over a 27 d period after flag leaf treatment with [14C]ethephon. Increasing amounts of [14C]ethephon metabolites (putative conjugates) were found in these organs over the same period. Breakdown of these conjugates, followed by ethylene release, probably accounts for elevated long‐term ethylene emanation from flag leaves and spikes. while ethephon was transported from the flag leaf to the spike, little transport was observed from the flag leaf to the base of the plant. Injections of ethephon into the base promoted tillering, and reduced elongation of the upper stem. Foliar ethephon treatments which reduced plant height also increased post‐treatment tillering. It is concluded that the tillering response following ethephon treatment does not include a modification of ethylene biosynthesis, and the amount of ethephon imported into the base is insufficient to be a direct signal for the tillering response. Induction of tiller growth may be a result of ethephon inhibition of upper stem elongation, rather than a direct response of the buds to ethephon.