Internal Reference Compounds Research Articles

The accuracy limit of chemical shift predictions for species in aqueous solution.

Interpreting NMR experiments benefits from first-principles predictions of chemical shifts. Reaching the accuracy limit of theory is relevant for unambiguous structural analysis and dissecting theoretical approximations. Since accurate chemical shift measurements are based on using internal reference compounds such as trimethylsilylpropanesulfonate (DSS), a detailed comparison of experimental with theoretical data requires simultaneous consideration of both target and reference species ensembles in the same solvent environment. Here we show that ab initio molecular dynamics simulations to generate liquid-state ensembles of target and reference compounds, including explicitly their short-range solvation environments and combined with quantum-mechanical solvation models, allows for predicting highly accurate 1H (∼0.1-0.5 ppm) and aliphatic 13C (∼1.5 ppm) chemical shifts for aqueous solutions of the model compounds trimethylamine N-oxide (TMAO) and N-methylacetamide (NMA), referenced to DSS without any system-specific adjustments. This encompasses the two peptide bond conformations of NMA identified by NMR. The results are used to derive a general-purpose guideline set for predictive NMR chemical shift calculations of NMA in the liquid state and to identify artifacts of force field models. Accurate predictions are only obtained if a sufficient number of explicit water molecules is included in the quantum-mechanical calculations, disproving a purely electrostatic model of the solvent effect on chemical shifts.

Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling in Arabidopsis thaliana Chloroplasts through Specific RelA/SpoT Homologues.

Magic spot nucleotides (p)ppGpp are important signaling molecules in bacteria and plants. In the latter, RelA-SpoT homologue (RSH) enzymes are responsible for (p)ppGpp turnover. Profiling of (p)ppGpp is more difficult in plants than in bacteria due to lower concentrations and more severe matrix effects. Here, we report that capillary electrophoresis mass spectrometry (CE-MS) can be deployed to study (p)ppGpp abundance and identity in Arabidopsis thaliana. This goal is achieved by combining a titanium dioxide extraction protocol and pre-spiking with chemically synthesized stable isotope-labeled internal reference compounds. The high sensitivity and separation efficiency of CE-MS enables monitoring of changes in (p)ppGpp levels in A. thaliana upon infection with the pathogen Pseudomonas syringae pv. tomato (PstDC3000). We observed a significant increase of ppGpp post infection that is also stimulated by the flagellin peptide flg22 only. This increase depends on functional flg22 receptor FLS2 and its interacting kinase BAK1 indicating that pathogen-associated molecular pattern (PAMP) receptor-mediated signaling controls ppGpp levels. Transcript analyses showed an upregulation of RSH2 upon flg22 treatment and both RSH2 and RSH3 after PstDC3000 infection. Arabidopsis mutants deficient in RSH2 and RSH3 activity display no ppGpp accumulation upon infection and flg22 treatment, supporting the involvement of these synthases in PAMP-triggered innate immune responses to pathogens within the chloroplast.

Use of high throughput ion channel profiling and statistical modeling to predict off-target arrhythmia risk - One pharma's experience and perspective

IntroductionThe use of high throughput patch clamp profiling to determine mixed ion channel-mediated arrhythmia risk was assessed using profiling data generated using proprietary internal and clinical reference compounds. We define the reproducibility of the platform and highlight inherent platform issues. The data generated was used to develop predictive models for cardiac arrhythmia risk, specifically Torsades de Pointes (TdP). MethodsA retrospective analysis was performed using profiling data generated over a 3-year period, including patch clamp data from hERG, Cav1.2, and Nav1.5 (peak/late), together with hERG binding. ResultsAssay reproducibility was robust over the 3-year period examined. High throughput hERG patch IC50 values correlated well with GLP-hERG data (Pearson = 0.87). A disconnect between hERG binding and patch was observed for ∼10% compounds and trended with passive cellular permeability. hERG and Cav1.2 potency did not correlate for proprietary compounds, with more potent hERG compounds showing selectivity versus Cav1.2. For clinical compounds where hERG and Cav1.2 activity was more balanced, an analysis of TdP risk versus hERG/Cav1.2 ratio demonstrated low TdP probability when the hERG/Cav1.2 potency ratios were < 1. Modeling of clinical compound data revealed a lack of impact of the Nav1.5 (late) current in predicting TdP. Moreover, models using hERG binding data (ROC AUC = 0.876) showed an improved ability to predict TdP risk versus hERG patch clamp (ROC AUC = 0.787). DiscussionThe data highlight the value of high throughput patch clamp data in the prediction of TdP risk, as well as some potential limitations with this approach.

Quantitative analysis of multi-components by single-marker: An effective method for the chemical characterization of wood vinegar

Wood vinegar is a complex mixture of organic compounds. QAMS is a relatively simple and cost-effective method which is used widely in recent years. In this study, quantitative analysis of multi-components by single-marker (QAMS) was applied to quantify the main volatile chemical components in wood vinegar for the first time. Using acetic acid as an internal reference compound, the relative correction factors of a further 19 chemical volatile components were obtained. 20 wood vinegar prepared from different lignocellulosic biomass, and at different pyrolysis temperatures, were analyzed by QAMS and the data compared with those obtained using an internal standard (phenethyl acetate) method. The results showed that the precision and repeatability of the QAMS method were good, and the responses obtained for each compound using GC/MS were linear over the corresponding concentration range (R2 ≥ 0.999). The average spike recoveries of 20 volatile compounds from wood vinegar ranged from 92.65% to 105.93% (RSD ＜ 7%, n = 6). There were no significant differences (P > 0.05) in the results obtained from the analysis of three batches of wood vinegar samples (RSD < 2%) using both methods. The volatile composition of samples obtained from wood vinegar prepared from the pyrolysis of different materials and at different temperatures were significantly different. The successful application of QAMS to the quantitative analysis of the composition of wood vinegar not only may provide a theoretical reference for further developments in the production, pyrolysis conditions, and modes of action for wood vinegar, but also provides ideas for the application of QAMS in other fields.

TMS is Superior to Residual CHCl3 for Use as the Internal Reference for Routine 1H NMR Spectra Recorded in CDCl3.

Tetramethylsilane was recommended for use as an internal reference compound in proton NMR spectroscopy over 60 years ago. However, it is a common practice that researchers reference the analyte chemical shifts to the residual proton resonance in the deuterated solvent in which the spectrum is recorded. Because CDCl3 is the most commonly used NMR solvent for routine analysis of organic compounds, the effect of various functional groups on the CHCl3 resonance is important. Described here are results that show why referencing spectra to TMS rather than CHCl3 in CDCl3 results in more accurate chemical shifts and should be the recommended practice. Simultaneous measurement of separate compartments of unperturbed CDCl3/TMS vis-à-vis CDCl3/TMS/solute solutions using a concentric tube arrangement was key. This study is reported in this venue because the audience/readership is quite appropriate and is, hopefully, both receptive to and appreciative of the guidance provided.

Changes in lipidomic profile by anti-retroviral treatment regimen: An ACTG 5257 ancillary study.

High cardiovascular disease risk in people living with HIV is partly attributed to antiretroviral therapy (ART). Lipid response to ART has been extensively studied, yet, little is known how small molecule lipids respond to Integrase inhibitor-based (INSTI-based) compared to Protease inhibitor-based (PI-based) ART regimens.Ancillary study to a phase 3, randomized, open-label trial [AIDS Clinical Trial Group A5257 Study] in treatment-naive HIV-infected patients randomized in a 1:1:1 ratio to receive ritonavir-boosted atazanavir (ATV/r), ritonavir-boosted darunavir (DRV/r) (both PI-based), or raltegravir with Tenofovir Disoproxil Fumarate-TDF plus emtricitabine (RAL, INSTI-based).We examined small molecule lipid response in a subcohort of 75 participants. Lipidomic assays of plasma samples collected pre- and post-ART treatment (48 weeks) were conducted using ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry. The effect of ART regimens was regressed on lipid species response adjusting for the baseline covariates (lipids, age, sex, race, CD4 level, BMI, and smoking). Results were validated in the Centers for AIDS Research Network of Integrated Clinical Systems study (N = 16).Out of 417 annotated lipids, glycerophospholipids (P = .007) and sphingolipids (P = .028) had a higher response to ATV/r and DRV/r compared to RAL. The lysophosphatidylcholine (LPCs(16:1),(17:1),(20:3)) and phosphophatidylcholine species (PCs(40:7),(38:4)) had an opposite response to RAL versus ATV/r in the discovery and validation cohort. The INSTI-based regimen had an opposite response of ceramide species ((d38:1), (d42:2)), PCs((35:2), (38:4)), phosphatidylethanolamines (PEs(38:4), (38:6)), and sphingomyelin(SMd38:1) species compared with the PI-based regimens. There were no differences observed between 2 PI-based regimens.We observed differences in response of small molecule lipid species by ART regimens in treatment-naive people living with HIV.

Selenate—An internal chemical shift standard for aqueous 77Se NMR spectroscopy

The 77 Se NMR spectra of selenate were studied under various circumstances, such as concentration, pH, temperature, ionic strength, and D2 O:H2 O ratio, in order to examine its potential as a water-soluble internal chemical shift standard. The performance of selenate as a chemical shift reference and that of other attempted ones from the literature (dimethyl selenide, tetramethylsilane/TMS, and 3-(trimethylsilyl)propane-1-sulfonate/DSS) was also explored. The uncertainty in the resulting chemical shift relative to the effective spectral width is comparable to that of DSS. Compared to the currently prevalent water-soluble external chemical shift reference, selenic acid solution, the properties of internal selenate are much more favorable in terms of ease of use. We have also demonstrated that selenate can be used in reducing media, which is inevitable for the analysis of selenol compounds. Thus, it can be stated that sodium selenate is a robust internal chemical shift reference in aqueous media for 77 Se NMR measurements; the chemical shift of this reference in a solution containing 5V/V% D2 O at 25°C and 0.15 mol·dm-3 ionic strength is 1048.65 ppm relative to 60 V/V% dimethyl selenide in CDCl3 and 1046.40 ppm relative to the 1 H signal of 0.03 V/V% TMS in CDCl3 . In summary, a water-soluble, selenium-containing internal chemical shift reference compound was introduced for 77 Se NMR measurements for the first time in the literature, and with the aforementioned results all previous 77 Se measurements can be converted to a unified scale defined by the International Union of Pure and Applied Chemistry.

Development and optimization of a cascade of screening assays for inhibitors of TRF2.

TRF2 is a telomere associated protein which plays an important role in telomere maintenance. Knockdown of TRF2 can cause chromosomal end to end fusions and induce DNA damage responses. TRF2 exerts its functions partially by recruiting a number of accessory proteins through its TRF homology domain (TRFH), therefore identification of small molecular compounds which can bind to the TRFH domain of TRF2 and block the interactions of TRF2 with its associated proteins is important to elucidate the molecular mechanism of these protein-protein interactions. Development of robust and sensitive screening and evaluation assays is critical to the identification of TRF2 inhibitors, in this paper we reported the development and optimization of a cascade of screening and binding affinity evaluation assays, including a competitive FP (Fluorescence Polarization) assay utilized in our previous research, and two novel label-free DSF (Differential Scanning Fluorescence) and BLI (Biolayer Interferometry) assays. A previously identified TRF2 inhibitor TRF2-27 was used as an internal reference compound and evaluated in all of these assays. According to the results, DSF assay is not suitable for TRF2 screening because of the low ΔTm, while the optimized labeled-free BLI assay was demonstrated to be an accurate and reproducible assay for TRF2 inhibitor screening and characterization.

Indoor and Outdoor BTEX in the Hospital and University Estates of Ouargla city, Algeria

Volatile organic compounds are ubiquitous atmospheric contaminants, affecting both indoor and outdoor environments. Attention is overall paid to toxicants, among which mono-aromatic species, in particular to benzene, toluene, ethylbenzene and xylenes are cumulatively indicated as BTEX. Mono-aromatic hydrocarbons were investigated in the air of Ouargla, Algeria, between 2014 and 2015. Measurements were conducted indoors and outdoors at the hospital and university estates of the city. BTEX were enriched from air by means of Analyst-I type diffusive sampling devices, which allowed continuous sampling over weeks. After collection, the samplers were spiked with bromobenzene (used as internal reference compound for analysis) and extracted with carbon disulfide directly in the vessel, then the analytes were characterized by means of capillary gas chromatography coupled with flame ionization detection. Total BTEXs ranged from ca. 41 to 131μg/m3; among them toluene (22–87μg/m3) was the most abundant congener. Benzene, ranging from 12 to 27μg/m3, always exceeded the limit value imposed by European normative for health preservation purposes (5μg/m3). At the hospital, the indoor/outdoor BTEX concentration ratio (I/O) exceeded all-the-time 1.0 (range: ∼1.5–2.0), which suggested the existence of internal sources for BTEX. This finding was confirmed by the analysis of concentration ratios between the BTEX compounds, which changed from outdoors to indoors as well as with the internal locations examined.

J‐difference‐edited MRS measures of γ‐aminobutyric acid before and after acute caffeine administration

The aim of this study was to investigate potential effects of acute caffeine intake on J-difference-edited MRS measures of the primary inhibitory neurotransmitter γ-aminobutyric acid (GABA). J-difference-edited Mescher-Garwood PRESS (MEGA-PRESS) and conventional PRESS data were acquired at 3T from voxels in the anterior cingulate and occipital area of the brain in 15 healthy subjects, before and after oral intake of a 200-mg caffeine dose. MEGA-PRESS data were analyzed with the MATLAB-based Gannet tool to estimate GABA+ macromolecule (GABA+) levels, while PRESS data were analyzed with LCModel to estimate levels of glutamate, glutamate+glutamine, N-acetylaspartate, and myo-inositol. All metabolites were quantified with respect to the internal reference compounds creatine and tissue water, and compared between the pre- and post-caffeine intake condition. For both MRS voxels, mean GABA+ estimates did not differ before and after caffeine intake. Slightly lower estimates of myo-inositol were observed after caffeine intake in both voxels. N-acetylaspartate, glutamate, and glutamate+glutamine did not show significant differences between conditions. Mean GABA+ estimates from J-difference-edited MRS in two different brain regions are not altered by acute oral administration of caffeine. These findings may increase subject recruitment efficiency for MRS studies.

Electrostatic Energetics of Bacillus subtilis Ribonuclease P Protein Determined by Nuclear Magnetic Resonance-Based Histidine pKa Measurements.

The pKa values of ionizable groups in proteins report the free energy of site-specific proton binding and provide a direct means of studying pH-dependent stability. We measured histidine pKa values (H3, H22, and H105) in the unfolded (U), intermediate (I), and sulfate-bound folded (F) states of RNase P protein, using an efficient and accurate nuclear magnetic resonance-monitored titration approach that utilizes internal reference compounds and a parametric fitting method. The three histidines in the sulfate-bound folded protein have pKa values depressed by 0.21 ± 0.01, 0.49 ± 0.01, and 1.00 ± 0.01 units, respectively, relative to that of the model compound N-acetyl-l-histidine methylamide. In the unliganded and unfolded protein, the pKa values are depressed relative to that of the model compound by 0.73 ± 0.02, 0.45 ± 0.02, and 0.68 ± 0.02 units, respectively. Above pH 5.5, H22 displays a separate resonance, which we have assigned to I, whose apparent pKa value is depressed by 1.03 ± 0.25 units, which is ∼0.5 units more than in either U or F. The depressed pKa values we observe are consistent with repulsive interactions between protonated histidine side chains and the net positive charge of the protein. However, the pKa differences between F and U are small for all three histidines, and they have little ionic strength dependence in F. Taken together, these observations suggest that unfavorable electrostatics alone do not account for the fact that RNase P protein is intrinsically unfolded in the absence of ligand. Multiple factors encoded in the P protein sequence account for its IUP property, which may play an important role in its function.

¹³C SPE MAS measurement of ligand concentration in compressible chromatographic beads.

A method for measuring the ligand concentration in heterogeneous materials like chromatography media is described. In this method, 13C single pulse excitation magic angle spinning NMR experiment with broadband 1H decoupling is used to determine the peak integrals for a butyl ligand in the spectrum of a dried chromatography medium. Within a carefully controlled protocol, those integrals compared with that of the internal reference compound dimethyl sulfone provide the required volume concentration with an accuracy of ca 2%. The effects of temperature, degree of hydration, and other experimental parameters are discussed. Copyright © 2015 The Authors. Magnetic Resonance in Chemistry published by John Wiley & Sons Ltd.

Synthesis of monomethyl 5,5′-dehydrodiferulic acid

Synthesis of the internal standard reference compound monomethyl 5,5′-dehydrodiferulic acid is described. The synthetic scheme relies on a selective monomethylation of the known compound 5,5′-dehydrodivanillin followed by elaboration into the dehydrodiferulic framework through a dual Horner–Emmons–Wadsworth reaction with methyl diethylphosphonoacetate. Saponification of the ester functionalities provided the target monomethyl 5,5′-dehydrodiferulic acid in 49% overall yield from 5,5′-dehydrodivanillin.

Propranolol Transport Across the Inner Blood–Retinal Barrier: Potential Involvement of a Novel Organic Cation Transporter

The influx transport of propranolol across the inner blood-retinal barrier (BRB) was investigated. In the in vivo analysis of carotid artery single-injection method, [(3) H]propranolol uptake by the retina was greater than that of an internal reference compound, and was reduced by several organic cations. In the in vitro uptake study, TR-iBRB2 cells, an in vitro model of the inner BRB, showed a time-, concentration-, pH- and temperature-dependent [(3) H]propranolol uptake, suggesting the involvement of a carrier-mediated transport process in the influx of propranolol across the inner BRB. In the inhibition study, various organic cations, including drugs and candidates for the treatment of the retinal diseases, inhibited the [(3) H]propranolol uptake by TR-iBRB2 cells with no significant effects by the substrates and inhibitors of well-characterized organic cation transporters, suggesting that the influx transport of propranolol is performed by a novel transporter at the inner BRB. An analysis of the relationship between the inhibitory effect and the lipophilicity of inhibitors suggests a lipophilicity-dependent inhibitory effect of amines on the [(3) H]propranolol uptake by TR-iBRB2 cells. These results showed that influx transport of propranolol across the inner BRB is performed by a carrier-mediated transport process, suggesting the involvement of a novel organic cation transporter.

Identification and removal of polycyclic aromatic hydrocarbons in wastewater treatment processes from coke production plants

Identification and removal of polycyclic aromatic hydrocarbons (PAHs) were investigated at two coke plants located in Shaoguan, Guangdong Province of China. Samples of raw coking wastewaters and wastewaters from subunits of a coke production plant were analyzed using gas chromatography-mass spectrometry (GC/MS) to provide a detailed chemical characterization of PAHs. The identification and characterization of PAH isomers was based on a positive match of mass spectral data of sample peaks with those for PAH isomers in mass spectra databases with electron impact ionization mass spectra and retention times of internal reference compounds. In total, 270 PAH compounds including numerous nitrogen, oxygen, and sulfur heteroatomic derivatives were positively identified for the first time. Quantitative analysis of target PAHs revealed that total PAH concentrations in coking wastewaters were in the range of 98.5 ± 8.9 to 216 ± 20.2μg/L, with 3-4-ring PAHs as dominant compounds. Calculation of daily PAH output from four plant subunits indicated that PAHs in the coking wastewater came mainly from ammonia stripping wastewater. Coking wastewater treatment processes played an important role in removing PAHs in coking wastewater, successfully removing 92% of the target compounds. However, 69 weakly polar compounds, including PAH isomers, were still discharged in the final effluent, producing 8.8 ± 2.7 to 31.9 ± 6.8g/day of PAHs with potential toxicity to environmental waters. The study of coking wastewater herein proposed can be used to better predict improvement of coke production facilities and treatment conditions according to the identification and removal of PAHs in the coke plant as well as to assess risks associated with continuous discharge of these contaminants to receiving waters.

Use of an Internal Reference for the Quantitative HPLC-UV Analysis of Solid-Phase Reactions: A Case Study of 2-Chlorotrityl Chloride Resin

Here we evaluated the use of internal reference compounds for the rapid assessment of reactions performed in solid-phase. An internal reference compound (commercially available) was bound to the resin, together with the substrate, and cleaved with the products after completion of the reaction. The peak area of the reference compound in the HPLC-UV chromatograms can be correlated directly with those of other compounds present in the reaction mixture, thereby allowing a quantitative interpretation of the chromatograms with respect to conversion and yield. The usefulness of this method was demonstrated by optimization of a protocol for the synthesis of proline-based tripeptides.

Accurate quantification of water–macromolecule exchange induced frequency shift: Effects of reference substance

Water-macromolecule exchange induces a bulk water frequency shift contributing to the contrast in phase imaging. For separating the effects of the water-macromolecule exchange and the macromolecule susceptibility, appropriate internal or external references are needed. In this study, two internal reference compounds, 2,2,3,3-tetradeuterio-3-trimethylsilyl-propionate (TMSP) and 1,4-dioxane, were used to study the macromolecule-dependent water frequency shift in a bovine serum albumin (BSA)-water system in detail. For TMSP, the water-macromolecule exchange shift depended on both the BSA and the reference concentration and stabilized to a value of 0.025 ppm/mM (298 K, TMSP concentrations > 30 mM). For dioxane, the dependency of the water-macromolecule exchange shift on the BSA concentration is independent of dioxane at low concentrations. The resulting shift was smaller (0.009 ppm/mM) when compared with using higher TMSP concentrations as reference. This discrepancy might be due to additional dioxane-water interactions. Measurements with an external chloroform reference in a coaxial geometry showed a shift of -0.013 ppm/mM resulting from the opposing effects of macromolecules in water exchange-induced shift and diamagnetic susceptibility shift. All these effects should be considered in the interpretation of tissue phase contrast. From the experimental data, the equilibrium binding constant between BSA and TMSP has been quantified to be K(d) = 1.3 ± 0.4, and the estimated number of interaction sites for BSA is 12.7 ± 2.6.

Measurement of absolute concentrations of individual compounds in metabolite mixtures by gradient-selective time-zero 1H-13C HSQC with two concentration references and fast maximum likelihood reconstruction analysis.

Time-zero 2D (13)C HSQC (HSQC(0)) spectroscopy offers advantages over traditional 2D NMR for quantitative analysis of solutions containing a mixture of compounds because the signal intensities are directly proportional to the concentrations of the constituents. The HSQC(0) spectrum is derived from a series of spectra collected with increasing repetition times within the basic HSQC block by extrapolating the repetition time to zero. Here we present an alternative approach to data collection, gradient-selective time-zero (1)H-(13)C HSQC(0) in combination with fast maximum likelihood reconstruction (FMLR) data analysis and the use of two concentration references for absolute concentration determination. Gradient-selective data acquisition results in cleaner spectra, and NMR data can be acquired in both constant-time and non-constant-time mode. Semiautomatic data analysis is supported by the FMLR approach, which is used to deconvolute the spectra and extract peak volumes. The peak volumes obtained from this analysis are converted to absolute concentrations by reference to the peak volumes of two internal reference compounds of known concentration: DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) at the low concentration limit (which also serves as chemical shift reference) and MES (2-(N-morpholino)ethanesulfonic acid) at the high concentration limit. The linear relationship between peak volumes and concentration is better defined with two references than with one, and the measured absolute concentrations of individual compounds in the mixture are more accurate. We compare results from semiautomated gsHSQC(0) with those obtained by the original manual phase-cycled HSQC(0) approach. The new approach is suitable for automatic metabolite profiling by simultaneous quantification of multiple metabolites in a complex mixture.

Caco‐2 Cells as a Model for Intestinal Absorption

The Caco-2 cell system, a well characterized intestinal in vitro model, makes it possible to evaluate the ability of chemicals to cross the intestinal barrier, as well as to study their transport mechanisms. Permeability values estimated with this model correlate well with human in vivo absorption data for many drugs and chemicals. As a consequence, the use of the Caco-2 cellular model as a permeability assay to predict oral absorption in humans is growing, and its importance is increasing as a screening tool in drug-discovery strategies for the prediction of intestinal drug permeability. Strict observance of the experimental conditions, coupled with careful control of the cell-culture procedures, are crucial for obtaining a meaningful correlation with in vivo data. Because of the intrinsic variability of this absorption model, it is important to highlight that each experimental system must be calibrated with internal reference compounds, to permit data sharing between different laboratories.

Quantitative determination of underivatized polyamines by using isotope dilution RP-LC–ESI-MS/MS

A rapid, sensitive and selective method using LC–MS/MS was developed and validated for the simultaneous quantitative determination of five polyamines N 1, N 12-diethylspermine (DESpm), N-ethylspermine (EtSpm), N 1-ethylspermidine (EtSpd), spermidine (Spd) and N 1-ethyl-1,3-diaminopropane (EtDAP) without any derivatization steps. The LC–MS/MS system was operated using the positive electrospray ionization (ESI) mode. The chromatographic separation only took 10 min and was performed on a reversed phase C18 column with 0.1% heptafluorobutyric acid as the ion-pairing agent and acetonitrile gradient. Stable, deuterium labelled internal reference compounds of the five analytes were included in the quantification. The lower limit of quantification for all of the five analytes was 0.03 μM and the method was linear for DESpm, EtSpd, Spd and EtDAP over the range of 0.03–60 μM and for EtSpm over the range of 0.03–30 μM. Correlation coefficients ( R 2) were always >0.995 for all the analytes. The precision of the overall method ranged from 0.2 to 9.7% as intra-day variability and from 0.9 to 6.8% as inter-day variability. The intra-day and inter-day accuracy of the assay ranged between 87.6–109.8% and 89.6–106.6%, respectively. The method has been applied successfully to quantify metabolites of DESpm as a substrate for recombinant human polyamine oxidase.