Application of stable carbon isotope analysis to the detection of 17beta-estradiol administration to cattle

The use of anabolic agents in food producing animals is prohibited within the EU since 1988 (96/22/EC directive). The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as far as no definitive method and non-ambiguous analytical criteria are available. The ability of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of 17beta-estradiol to bovine has been investigated in this paper. By comparison of 13C/12C isotopic ratio of main urinary estradiol metabolite, i.e. 17alpha-estradiol, with two endogenous reference compounds (ERCs), i.e. dehydroepiandrosterone (DHEA) and 5-androstene-3beta,17alpha-diol, the differentiation of estradiol metabolite origin, either endogenous or exogenous, has been proved to be achievable. After treatment, the delta(13)C(VPDB)-values of 17alpha-estradiol reached -27 per thousand to -29 per thousand, whereas delta13CVPDB-values of DHEA remained between -13 per thousand and -20 per thousand depending on the diet, maize and grass, respectively. A significant difference of delta13CVPDB between ERCs and 17alpha-estradiol was measurable over a period of 2 weeks after estradiol ester administration to the animal.     

Development and application of stable carbon isotope analysis to the detection of cortisol administration in cattle

The use of anabolic agents in food-producing animals has been prohibited within the EU since 1988. The control of the illegal use of natural steroid hormones in cattle is still an exciting analytical challenge as no definitive method and nonambiguous analytical criteria are available. We have used gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to demonstrate the administration of cortisol to cattle. The method consisted of an efficient combination between OASIS HLB solid-phase extraction (SPE), oxidation, SiOH SPE and semi-preparative high-performance liquid chromatography (HPLC) for glucocorticoid purification. By comparison of the (13)C/(12)C isotopic ratio of the oxidised product of cortisol, i.e. 5 beta-androstane-3,11,17-trione (5 beta AAT), with an endogenous reference compound (ERC), dehydroepiandrosterone (DHEA), the differentiation of cortisol metabolite origin, either endogenous or exogenous, has been achieved. After treatment of an animal, the delta(13)C(VPDB) values of 5 beta AAT reached -30 to -32 per thousand, whereas the delta(13)C(VPDB) values of DHEA remained at -25 per thousand. A significant difference in the delta(13)C(VPDB) values between DHEA and 5 beta AAT was measurable over a period of 3 days after a single administration of cortisol to the animal.     

A new method for stable lead isotope extraction from seawater

A new technique for stable lead (Pb) isotope extraction from seawater is established using Toyopearl AF-Chelate 650 M^® resin (Tosoh Bioscience LLC). This new method is advantageous because it is semi-automated and relatively fast; in addition it introduces a relatively low blank by minimizing the volume of chemicals used in the extraction. Subsequent analyses by HR ICP-MS have a good relative external precision (2σ) of 3.5‰ for ²⁰⁶Pb/²⁰⁷Pb, while analyses by MC-ICP-MS have a better relative external precision of 0.6‰. However, Pb sample concentrations limit MC-ICP-MS analyses to ²⁰⁶Pb, ²⁰⁷Pb, and ²⁰⁸Pb. The method was validated by processing the common Pb isotope reference material NIST SRM-981 and several GEOTRACES intercalibration samples, followed by analyses by HR ICP-MS, all of which showed good agreement with previously reported values.     

Distribution and metabolism of selenohomolanthionine labeled with a stable isotope

The distribution and metabolism of selenohomolanthionine (4,4′-selenobis[2-aminobutanoic acid], SeHLan), a newly identified selenoamino acid in selenized Japanese pungent radish, were evaluated by administering ⁷⁷Se-labeled SeHLan at a dose of 25 μg/kg body weight in rats. Exogenous ⁷⁷Se of SeHLan was preferably distributed to the kidneys and remained in the intact form for up to 6 h after dosing. The accumulation in the kidneys is one of the specific characteristics of SeHLan, differing from other selenoamino acids, such as selenomethionine and Se-methylselenocysteine, which preferably accumulate in the pancreas. The intact form of SeHLan was detected in the serum and kidney supernatant but not in the urine, suggesting that the amount of exogenous Se that was distributed to the kidneys was within metabolic capacity. Indeed, the exogenous Se was converted into two urinary metabolites, Se-methylseleno-N-acetyl-galactosamine and trimethylselenonium. Exogenous Se was also detected in several selenoproteins, including selenoprotein P and extracellular glutathione peroxidase. SeHLan is expected to be a potential supplemental source of Se because its distribution differs from that of selenomethionine and Se-methylselenocysteine.     

Precise proteomic identification using mass spectrometry coupled with stable isotope labeling

Stable isotope labeling (SIL) can assist mass spectrometry to improve its specificity and throughput in protein identification, de novo sequencing, and characterization of post translational modifications. This Education article summarizes the unique characteristics of stable isotope-assisted mass spectrometry for accurate protein identification without requirements for ultrahigh mass accuracy. Some applications are discussed here to demonstrate the general experimental procedures, data interpretation, and the improvements in accuracy and throughput compared to the use of mass spectrometry alone.     

A technique for estimating contamination tolerance limits for stable isotope ratio determinations

A model of the interaction between the precision of an isotope ion signal measurement and the accuracy of an isotope ratio determination was developed and used to derive the equations required to calculate the maximum tolerable amount of contamination for stable isotope ratio determinations. Comparison of the calculated tolerance limits and the blank estimated amount of contaminant will establish whether or not a correction for the contribution of the contaminant to the gross signals will be required. The 1000:1 sample-to-contaminant concentration tolerance limit used in stable isotope ratio plasma mass spectrometry will, in some situations, underestimate the contamination error compared to the model calculations.Derivation of the limit equations required an empirically determined relation between the signal strength and the signal's relative standard deviation (signal noise function). A single hyperbolic signal noise function was used to describe the behaviour of isotope ion signals of Ni, Cu, Tl and Pb measured using a plasma source double focusing magnetic sector mass spectrometer. The derivation could be extended to accommodate different signal noise functions.     

Detection of honey adulterations with sugar syrups by stable isotope mass spectrometry

A procedure is proposed for determining the quality of honey by measuring the isotope composition of carbon in the initial honey samples and their protein fractions by mass spectrometry. Seventeen samples of honey harvested in 2014 in various regions of Russia were investigated to identify falsifications with sugars or invert syrups. Using the data on the isotope ratios of δ¹³C of initial honey samples and protein fractions, the degree of adulteration of the test honey samples was determined. Concentration of sugar was used as a criterion of adulteration. According to the data obtained, in two samples of honey, the difference between the δ¹³C values in the protein fraction and the original honey was more than 1‰, indicating the dilution of these honey samples with cane sugar by more than 7%. It is shown that the isotope composition of honey is not only informative for detecting the adulteration of honey, but also can serve as a kind of marker for the geographical origin of honey.     

A versatile method for stable carbon isotope analysis of carbohydrates by high-performance liquid chromatography/isotope ratio mass spectrometry

We have developed a method to analyze stable carbon isotope ((13)C/(12)C) ratios in a variety of carbohydrates using high-performance liquid chromatography/isotope ratio mass spectrometry (HPLC/IRMS). The chromatography is based on strong anion-exchange columns with low strength NaOH eluents. An eluent concentration of 1 mM resulted in low background signals and good separation of most of the typical plant neutral carbohydrates. We also show that more strongly bound carbohydrates such as acidic carbohydrates can be separated by inclusion of NO(3) (-) as an inorganic pusher ion in the eluent. Analyses of neutral carbohydrate concentrations and their stable carbon isotope ratios are shown for plant materials and marine sediment samples both at natural abundance and for (13)C-enriched samples. The main advantage of HPLC/IRMS analysis over traditional gas chromatography based methods is that no derivatization is needed resulting in simple sample treatment and improved accuracy and reproducibility.     

稳定同位素稀释技术在人体肝脏维生素A储备量检测中的应用

建立了稳定同位素稀释技术结合高效液相色谱-气相色谱/负化学电离源质谱法(HPLC-GC/NCI/MS)测定人体肝脏维生素A(VA)储备量的方法。志愿者在口服氘标记的视黄醇醋酸酯(²H₈-RAC)后,经21天的稳定期,采集血样,分离血清。血清经正己烷提取,高效液相色谱分离纯化后,氮气吹干供进一步衍生化,衍生化产物用GC/NCI/MS检测,获得标记与未标记VA的丰度比,最后利用Furr-Olson公式计算VA肝脏储备量。在优化的条件下,血清样本中VA的回收率大于85%,精密度(RSD, n=6)小于10%,定量限为26.4 μ g/L,基本满足口服1 mg ²H₈-RAC后标记与未标记VA的检测要求。相比国内现行的人体VA评价方法,该方法能更客观地反映人体VA营养水平。该项测定技术与VA干预实验相结合,可获得某一人群维持体内VA稳定储备水平的膳食摄入量水平。同时,稳定同位素示踪技术的运用对促进经口摄入的VA源在体内生物转化效率的研究也起着重要作用。     

A simplified method of preparing phosphoric acid for stable isotope analyses of carbonates

In order to produce CO₂ for stable isotope analyses (δ¹⁸O and δ¹³C), carbonate samples are commonly digested in phosphoric acid. The acid recipe here presented is based on phase shifting crystalline orthophosphoric acid of pro‐analysis quality to a liquid state through heating, followed by pre‐vacuum treatment during a start‐up procedure before mass analyses for common acid bath preparation, or adding a small amount of phosphoric pentoxide for single drop equipments, respectively. This methodology results in a final acid concentration of 104%. Copyright © 2005 John Wiley & Sons, Ltd.     

A minimal proteinlike lattice model: an alpha-helix motif

A simple protein model of a four-helix bundle motif on a face-centered cubic lattice has been studied. Total energy of a conformation includes attractive interactions between hydrophobic residues, repulsive interactions between hydrophobic and polar residues, and a potential that favors helical turns. Using replica exchange Monte Carlo simulations we have estimated a set of parameters for which the native structure is a global minimum of conformational energy. Then we have shown that all the above types of interactions are necessary to guarantee the cooperativity of folding transition and to satisfy the thermodynamic hypothesis.     

A model for isotope separation via molecular multiphoton photodissociation

Multiphoton photofragmentation of an “isolated” molecule on the lowest potential surface is considered in terms of a truncated anharmonic oscillator driven by a pulsed, intense laser field. Intramolecular vibrational relaxation and indirect photodissociation are accounted for by assignment of decay widths to the appropriate levels. The effective-hamiltonian formalism is applied to derive explicit expressions for the photofragmentation yield and for the isotopic separation factor.