Nicotine,acetanilide and urea multi‐level 2H‐, 13C‐ and 15N‐abundance reference materials for continuous‐flow isotope ratio mass spectrometry

Accurate determinations of stable isotope ratios require a calibration using at least two reference materials with different isotopic compositions to anchor the isotopic scale and compensate for differences in machine slope. Ideally, the δ values of these reference materials should bracket the isotopic range of samples with unknown δ values. While the practice of analyzing two isotopically distinct reference materials is common for water (VSMOW‐SLAP) and carbonates (NBS 19 and L‐SVEC), the lack of widely available organic reference materials with distinct isotopic composition has hindered the practice when analyzing organic materials by elemental analysis/isotope ratio mass spectrometry (EA‐IRMS). At present only L‐glutamic acids USGS40 and USGS41 satisfy these requirements for δ¹³C and δ¹⁵N, with the limitation that L‐glutamic acid is not suitable for analysis by gas chromatography (GC). We describe the development and quality testing of (i) four nicotine laboratory reference materials for on‐line (i.e. continuous flow) hydrogen reductive gas chromatography‐isotope ratio mass‐spectrometry (GC‐IRMS), (ii) five nicotines for oxidative C, N gas chromatography‐combustion‐isotope ratio mass‐spectrometry (GC‐C‐IRMS, or GC‐IRMS), and (iii) also three acetanilide and three urea reference materials for on‐line oxidative EA‐IRMS for C and N. Isotopic off‐line calibration against international stable isotope measurement standards at Indiana University adhered to the ‘principle of identical treatment’. The new reference materials cover the following isotopic ranges: δ²H_nicotine ?162 to ?45‰, δ¹³C_nicotine ?30.05 to +7.72‰, δ¹⁵N_nicotine ?6.03 to +33.62‰; δ¹⁵N_acetanilide +1.18 to +40.57‰; δ¹³C_urea ?34.13 to +11.71‰, δ¹⁵N_urea +0.26 to +40.61‰ (recommended δ values refer to calibration with NBS 19, L‐SVEC, IAEA‐N‐1, and IAEA‐N‐2). Nicotines fill a gap as the first organic nitrogen stable isotope reference materials for GC‐IRMS that are available with different δ¹⁵N values. Comparative δ¹³C and δ¹⁵N on‐line EA‐IRMS data from 14 volunteering laboratories document the usefulness and reliability of acetanilides and ureas as EA‐IRMS reference materials. Published in 2009 by John Wiley & Sons, Ltd.     

Quantification of peptides using N‐terminal isotope coding and C‐terminal derivatization for sensitive analysis by micro liquid chromatography‐tandem mass spectrometry

Stable isotope‐coding coupled with mass spectrometry is a popular method for quantitative proteomics and peptide quantification. However, the efficiency of the derivatization reaction at a particular functional group, especially in complex structures, can affect accuracy. Here, we present a dual functional‐group derivatization of bioactive peptides followed by micro liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). By separating the sensitivity‐enhancement and isotope‐coding derivatization reactions, suitable chemistries can be chosen. The peptide amino groups were reductively alkylated with acetaldehyde or acetaldehyde‐d₄ to afford N‐alkylated products with different masses. This process is simple, quick and high‐yield, and accurate comparative analysis can be achieved for the mass‐differentiated peptides. Then, the carboxyl groups were derivatized with 1‐(2‐pyrimidinyl)piperazine to increase MS/MS sensitivity. Angiotensins I–IV, bradykinin and neurotensin were analyzed after online solid phase extraction by micro LC‐MS/MS. In all instances, a greater than 17‐fold increase in sensitivity was achieved, compared with the analyses of the underivatized peptides. Furthermore, the values obtained from the present method were in agreement with the result from isotope dilution quantification using isotopically labeled angiotensin I [Asp‐Arg‐(Val‐d₈)‐Tyr‐Ile‐His‐Pro‐(Phe‐d₈)‐His‐Leu]. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous quantification of N‐butylthiophosphoric triamide and dicyandiamide in urea formulation by liquid chromatography with tandem mass spectrometry

A new liquid chromatography with tandem mass spectrometry method employing a mixed‐mode zwitterionic stationary phase was developed for simultaneous determination of urease inhibitor (N‐butylthiophosphoric triamide) and nitrification inhibitor (dicyandiamide) in urea fertilizer. Molecular modeling based on density functional theory calculations was employed to provide an insight into the interaction mechanism of urea, dicyandiamide, and N‐butylthiophosphoric triamide with zwitterionic stationary phase in chromatographic separation system. The detection of analytes was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode using positive electrospray ionization. The ion transitions monitored were m/z 85→68 for dicyandiamide and m/z 168.2→74 for N‐butylthiophosphoric triamide, respectively. The standard calibration curves of dicyandiamide and N‐butylthiophosphoric triamide were linear over the range of 1.0 ? 15 ppm (coefficient of determination = 0.9984), 0.05 ? 1 ppm (coefficient of determination = 0.9995), with limit of detection of 25 and 5 ppb, respectively. The recoveries of low, middle, and high concentrations were from 96.7 to 105.8% for N‐butylthiophosphoric triamide and 94.4 to 105.8% for dicyandiamide with accuracy (relative error %) of ≤5.8% and ≤5.8%, the precision (coefficients of variation) was ≤2.0% and ≤2.9%, respectively. The validated method was successfully applied on real urea samples to determine N‐butylthiophosphoric triamide and dicyandiamide simultaneously.     

C‐phosphorylation of N‐arylpyrroles

Phosphorylation of N‐arylpyrroles with phosphorus tribromide proceeds regioselectively at the position 2 of the heterocyclic system. A 2‐to‐3 migration of the dibromophosphino group has been discovered, with its ease depending on the electronic nature of a substituent on the phenyl ring, solvent polarity, and the presence of pyridine hydrobromide in the reaction mixture. Further phosphorylation of 2‐ and 3‐monophosphorylated N‐arylpyrroles regioselectively involves the respective positions 4 and 5 of the heterocycle and is governed by the electron‐acceptor effect of the phosphorus‐containing substituent. © 2002 Wiley Periodicals, Inc. Heteroatom Chem 13:223–228, 2002; Published online in Wiley Interscience (www.interscience.wiley.com). DOI 10.1002/hc.10012     

1H‐, 13C‐, and 15N‐NMR chemical shifts for selected glucosides and ribosides of aromatic cytokinins

The ¹H and ¹³C NMR resonances of 16 purine glucosides were assigned by a combination of one‐ and two‐dimensional NMR experiments, including gs‐COSY, gs‐HSQC, and gs‐HMBC, in order to characterize the effect of substituent and the position of glucose unit on the NMR chemical shifts. In addition, ¹⁵N NMR chemical shifts for selected derivatives were investigated by using ¹H? ¹⁵N chemical shift correlation techniques. To map the influence of sugar moiety on the directly bonded nitrogen atom, selected N⁹‐glucosides and their ribose analogs were compared. Characteristic long‐range ¹H? ¹⁵N coupling constants, measured by using ¹H? ¹⁵N gradient‐selected single‐quantum multiple bond correlation (GSQMBC), are also reported and discussed. All compounds investigated here belong to cytokinins, an important group of plant hormones. Copyright © 2010 John Wiley & Sons, Ltd.     

Simultaneous analysis of 13C‐glutathione as its dimeric form GSSG and its precursor [1‐13C]glycine using liquid chromatography/isotope ratio mass spectrometry

Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine ¹³C‐glutathione as its dimeric form (GSSG) and its precursor [1‐¹³C]glycine in a small volume of erythrocytes in one single analysis. After having transformed ¹³C‐glutathione into its dimeric form GSSG, we determined both the intra‐erythrocytic concentrations and the ¹³C‐isotopic enrichment of GSSG and glycine in 150 µL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of µmol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 µmol/mL. The ¹³C‐isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3‰) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous characterization of pyrrolizidine alkaloids and N‐oxides in Gynura segetum by liquid chromatography/ion trap mass spectrometry

Gynura segetum (Lour.) Merr. (Jusanqi) is a traditional herbal product used for hemostasis and detumescence in Chinese folk medicine. However, its hepatic toxicity should not be ignored. In this study, pyrrolizidine alkaloids (PAs) and their corresponding N‐oxides (PANOs) were extracted from the whole plant of G. segetum and analyzed by high‐performance liquid chromatography coupled to ion trap mass spectrometry (ITMS). Identification of eluted peaks as PANOs was indicated by virtue of their MS and MSⁿ analysis, in addition to the [M+H]⁺ adduct ion, characteristically showed a significant (usually 100% abundance) dimer adduct [2M+H]⁺ that is not observed in the MS of the parent PAs. A total of 20 compounds were identified or tentatively characterized based on their mass spectra and possible biosynthetic pathways, of which three PAs and one PANO, namely seneciphylline, senecionine, seneciphylline and seneciphyllinine N‐oxide, were unequivocally characterized, while other PAs and PANOs were tentatively assigned. Sixteen constituents were reported for the first time from G. segetum and tetrahydrosenecionine has not been previously reported as a natural product. Our results are the first comprehensive analysis of PAs and PANOs in G. segetum constituents and will be helpful for the quality control of the herb of G. segetum and its related preparations. Copyright © 2008 John Wiley & Sons, Ltd.     

Stereochemical differentiation of C‐7 hydroxyltaxane isomers by electrospray ionization mass spectrometry

In this study, different electrospray ionization mass spectrometric (ESI‐MS) methods were utilized to analyze several pairs of taxane stereoisomers including paclitaxel and 7‐epi‐paclitaxel. Both ESI‐MS and tandem mass spectrometry (MS/MS) techniques provided stereochemically dependent mass spectra in negative‐ion mode, and all studied stereoisomers could be easily distinguished based on their characteristic ions or distinct fragmentation patterns. MS/MS experiments for several taxane analogues at various collision energies were performed to elucidate potential dissociation pathways. The gas‐phase deprotonation potentials were also calculated to estimate the most thermodynamically favorable deprotonation site using DFT B3LYP/6‐31G(d). The results of the theoretical studies agreed well with the fragmentation patterns of paclitaxel and 7‐epi‐paclitaxel observed from MS/MS experiments. In addition, it was found that liquid chromatography (LC)/ESI‐MS was a useful and sensitive technique for assignment of C‐7 taxane stereoisomers from realistic samples. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of four thiophenethylamine designer drugs (2C‐T‐4, 2C‐T‐8, 2C‐T‐13, 2C‐T‐17) in human urine by capillary electrophoresis/mass spectrometry

An analytical procedure for the simultaneous determination in human urine of four thiophenethylamine designer drugs (2C‐T series) is reported. The quantitative analysis was performed by capillary electrophoresis with mass spectrometric detection (CE/MS), using 2,5‐dimethoxy‐4‐methylthiophenethylamine‐D₄ (2C‐T‐D₄) as internal standard. In order to minimize interferences with matrix components and to preconcentrate target analytes, solid‐phase extraction (SPE) was introduced in the method as a clean‐up step. The method was validated according to international guidelines. The data for accuracy and precision were within required limits. Calibration curves were generated over the range from 10 to 500 ng mL^?1 and correlation coefficients always exceeded 0.997. The method was demonstrated to be specific, sensitive, and reliable for the analysis of these derivatives in urine samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Mapping site‐specific protein N‐glycosylations through liquid chromatography/mass spectrometry and targeted tandem mass spectrometry

Glycosylation is one of the most common posttranslational modifications (PTMs) of proteins, the characterization of which is commonly achieved through proteomic protocol, involving trypsin digestion followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, it is often not possible to characterize all glycopeptides in a complex sample because of the high complexity of glycoproteomic samples, and the relative lower abundances of glycopeptides in comparison to the unmodified peptides. We present here a targeted MS/MS analysis approach, which utilizes a previously developed computational tool, GlyPID, to guide multiple experiments, thus permitting a complete characterization of all N‐glycosylation sites of glycoproteins present in a complex sample. We have tested our approach using model glycoproteins analyzed by high‐resolution LTQ‐FT MS. The results demonstrate a potential use of our method for a high‐throughput characterization of complex mixtures of glycosylated proteins. Copyright © 2010 John Wiley & Sons, Ltd.