Determination of l-cysteine origin on the basis of its δ15N values.

The majority of l-cysteine is obtained industrially by hydrolysis of animal materials, such as poultry feathers. Despite widespread belief, there is little evidence that human hair is used as a source material and its use is explicitly banned in the European Union (2000/63/EC decision). We developed an isotope ratio mass spectrometric (EA-IRMS) method to determine carbon and nitrogen isotopic ratio in cysteine preparations and related compounds, e.g. cystine and carbocysteine. A threshold relying on the 15N/14N was established to differentiate between hair and feathers; a value below 6.6‰ indicates a poultry feathers origin. Global uncertainty of measurement was found to be 0.1‰ for δ15N (sample size of 0.5-1.8 mg).

Analysis of glucuronide and sulfate steroids in urine by ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry.

Profiling conjugated urinary steroids to detect anabolic-steroid misuse is recognized as an efficient analytical strategy in both chemical-food-safety and anti-doping fields. The relevance and robustness of such profiling rely on the analysis of glucuronide and sulfate steroids, which is expected to have properties including accuracy, specificity, sensitivity, and, if possible, rapidity. In this context, the ability of ultra-high-performance supercritical-fluid chromatography (UHPSFC) hyphenated tandem mass spectrometry (MS-MS) to provide reliable and accurate phase II analysis of steroids was assessed. Four stationary phases with sub-2 µm particles (BEH, BEH 2-ethyl-pyridine, HSS C18 SB, and CSH fluorophenyl) were screened for their capacity to separate several conjugated steroid isomers. Analytical conditions including stationary phase, modifier composition and percentage, back pressure, column temperature, and composition and flow rate of make-up solvent were investigated to improve the separation and/or the sensitivity. Thus, an analytical procedure enabling the analysis of eight glucuronide and 12 sulfate steroids by two different methods in 12 and 15 min, respectively, was optimized. The two procedures were evaluated, and UHPSFC-MS-MS analysis revealed its ability to provide sensitive (limits of quantification: 0.1 ng mL(-1) and 0.5 ng mL(-1) for sulfate and glucuronide steroids, respectively) and reliable quantitative performance (R(2) > 0.995, RSD < 20%, and bias < 30%) through the use of suitable labeled internal standards. Comparison with UHPLC-MS-MS was performed, and UHPSFC-MS-MS obtained better performance in terms of sensitivity. Finally, as a proof of concept, this so-called green technology was used in a chemical-food-safety context to profile steroid conjugates in urine samples from bovines treated with estradiol. Graphical Abstract Glucuronide and sulfate steroids analysis in urine by ultra-high performance supercritical fluid chromatography hyphenated tandem mass spectrometry.

Direct analysis in real time - high resolution mass spectrometry (DART-HRMS): a high throughput strategy for identification and quantification of anabolic steroid esters.

High throughput screening is essential for doping, forensic, and food safety laboratories. While hyphenated chromatography-mass spectrometry (MS) remains the approach of choice, recent ambient MS techniques, such as direct analysis in real time (DART), offer more rapid and more versatile strategies and thus gain in popularity. In this study, the potential of DART hyphenated with Orbitrap-MS for fast identification and quantification of 21 anabolic steroid esters has been evaluated. Direct analysis in high resolution scan mode allowed steroid esters screening by accurate mass measurement (Resolution = 60 000 and mass error < 3 ppm). Steroid esters identification was further supported by collision-induced dissociation (CID) experiments through the generation of two additional ions. Moreover, the use of labelled internal standards allowed quantitative data to be recovered based on isotopic dilution approach. Linearity (R(2) > 0.99), dynamic range (from 1 to 1000 ng mL(-1) ), bias (<10%), sensitivity (1 ng mL(-1) ), repeatability and reproducibility (RSD < 20%) were evaluated as similar to those obtained with hyphenated chromatography-mass spectrometry techniques. This innovative high throughput approach was successfully applied for the characterization of oily commercial preparations, and thus fits the needs of the competent authorities in the fight against forbidden or counterfeited substances.

High throughput identification and quantification of anabolic steroid esters by atmospheric solids analysis probe mass spectrometry for efficient screening of drug preparations.

Recent developments in ambient mass spectrometry (AMS), such as atmospheric solids analysis probe (ASAP) mass spectrometry, open a whole new range of possibilities to screen for drug preparations. In this study, the potential of ASAP for the rapid identification and quantification of anabolic steroid esters has been evaluated. These compounds are known to be used both in human and in food producing animals to enhance performances and to improve the rate of growth, respectively. Using a triple quadrupole (QqQ) MS instrument, mechanism of ionization and fragmentation in both positive and negative mode were studied for a range of 21 selected steroid esters (based on testosterone, estradiol, nandrolone, and boldenone) which highlighted common neutral mass loss of 96.1, thus allowing rapid screening in minutes to reveal steroid ester presence with minimal sample preparation. Ester identification is further achieved through an efficient 2 min workflow on a QqQ MS instrument. Moreover, the use of isotope labeled internal standards permitted the quantification of the corresponding steroid esters in selected reaction monitoring (SRM) mode, for the first time in ASAP. This approach was successfully applied for characterization of oily commercial preparations. These results open new perspectives in hormone (and drug) rapid analysis by ASAP-MS in the near future.

Germination stimulants of Phelipanche ramosa in the rhizosphere of Brassica napus are derived from the glucosinolate pathway.

Phelipanche ramosa is a major parasitic weed of Brassica napus. The first step in a host-parasitic plant interaction is stimulation of parasite seed germination by compounds released from host roots. However, germination stimulants produced by B. napus have not been identified yet. In this study, we characterized the germination stimulants that accumulate in B. napus roots and are released into the rhizosphere. Eight glucosinolate-breakdown products were identified and quantified in B. napus roots by gas chromatography-mass spectrometry. Two (3-phenylpropanenitrile and 2-phenylethyl isothiocyanate [2-PEITC]) were identified in the B. napus rhizosphere. Among glucosinolate-breakdown products, P. ramosa germination was strongly and specifically triggered by isothiocyanates, indicating that 2-PEITC, in particular, plays a key role in the B. napus-P. ramosa interaction. Known strigolactones were not detected by ultraperformance liquid chromatography-tandem mass spectrometry, and seed of Phelipanche and Orobanche spp. that respond to strigolactones but not to isothiocyanates did not germinate in the rhizosphere of B. napus. Furthermore, both wild-type and strigolactone biosynthesis mutants of Arabidopsis thaliana Atccd7 and Atccd8 induced similar levels of P. ramosa seed germination, suggesting that compounds other than strigolactone function as germination stimulants for P. ramosa in other Brassicaceae spp. Our results open perspectives on the high adaptation potential of root-parasitic plants under host-driven selection pressures.