Liquid chromatography/tandem mass spectrometry for the identification and determination of trichothecenes in maize

A reliable, sensitive and selective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed to determine four trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X and 3-acetyldeoxynivalenol) in maize. Sample preparation was performed by extracting the analytes with a mixture of acetonitrile and water, followed by a solid-phase extraction with Carbograph-4 cartridges as the purification step. For the LC/MS/MS analysis two interfacing systems, Turbo IonSpray (TISP) and atmospheric pressure chemical ionization (APCI), were compared in both negative and positive ion modes. LC and MS parameters were optimized to achieve better results and sensitivity. The effect of mobile phase modifiers such as ammonium acetate and formic acid on the ionization yield was also evaluated. The best results were obtained using the electrospray ionization (ESI) interface in negative ion mode and the multiple reaction monitoring mode (MRM) for the quantitation. The detection limits ranged between 10 ng/g for fusarenon X and 1.5 ng/g for deoxynivalenol. A linear working range was achieved with a standard deviation between 3 and 10% and recovery rates from the maize samples above 81%. The procedure was applied to the analysis of a set of maize samples collected from farms located in different areas of northern and central Italy. The investigated samples turned out to be contaminated primarily with deoxynivalenol and, to a minor extent, with its derivatives.     

Incorporation of methamphetamine and amphetamine in human hair following controlled oral methamphetamine administration

Background

Although hair testing is well established for the assessment of past drug exposure, uncertainties persist about mechanisms of drug incorporation into hair and interpretation of results. The aim of this study was to administer methamphetamine (MAMP) under controlled conditions as a model drug to investigate drug incorporation into human hair.

Material and methods

Seven volunteers with a history of stimulant use received 4 × 10 mg (low) doses of sustained release S-(+)-MAMP HCl within 1 week, with weekly head hair samples collected by shaving. 3 weeks later, 4 of them received 4 × 20 mg (high) doses. After extensive isopropanol/phosphate buffer washing of the hair, MAMP and its metabolite amphetamine (AMP) concentrations were determined in all weekly hair samples by LC–MS–MS in selected reaction monitoring mode with the undeca- and deca-deuterated drugs, respectively, as internal standards (LLOQ, 0.005 ng mg⁻¹).

Results

MAMP T_max occurred from 1 to 2 weeks after both doses, with C_max ranging from 0.6 to 3.5 ng mg⁻¹ after the low and 1.2 to 5.3 ng mg⁻¹ after the high MAMP doses. AMP C_max in hair was 0.1–0.3 ng mg⁻¹ and 0.2–0.5 ng mg⁻¹, respectively, for low and high doses. Highly dose-related concentrations within subjects, but large variability between subjects were observed. MAMP concentrations were above the 0.2 ng mg⁻¹ cut-off for at least 2 weeks following administration of both low and high doses. The overall AMP/MAMP ratio ranged from 0.07 to 0.37 with a mean value of 0.15 ± 0.07, and a median of 0.13. The percentage of MAMP and AMP removed with the washing procedure decreased with time after administration. A strong correlation was found between area under the curve of MAMP (r² = 0.90, p = 0.00) and AMP (r² = 0.94, p = 0.00) concentrations calculated for the 3-week period following administration and the total melanin concentration in hair. Significant correlations were observed also between C_max and melanin.

Conclusions

This study demonstrated that despite large inter-individual differences, the incorporation of MAMP and AMP into hair is dose-related with much of the observed scatter of MAMP and AMP concentrations explained by melanin concentration in hair.     

Development of a chemiluminescent enzyme immunoassay for urinary 1-hydroxypyrene

A chemiluminescent enzyme-immunoassay for urinary 1-hydroxypyrene has been developed and optimized. The enzymatic activity of horseradish peroxidase-labeled tracer was measured with an enhanced chemiluminescent system and the results were compared with those from conventional colorimetric detection. The method fulfilled all the requirements of accuracy and precision and the detection limit was 0.001 pmol/well, which enabled analysis in less than 1 microL urine. Subjects working in the center of Bologna who were exposed daily to vehicular exhaust gas were studied. Their urinary 1-hydroxypyrene concentrations were compared with the levels of benzo( a)pyrene in air particulate matter. Urinary 1-hydroxypyrene, which ranged from 0.5 to 10 nmol L(-1), correlated poorly with the concentration of benzo( a)pyrene in air particulate matter, which ranged from 5 to 140 ng m(-3). No significant effect of vehicle exhaust gas exposure was observed among the different groups of subjects working in different areas of the town. Thus, at a relatively low level of exposure 1-hydroxypyrene does not seem to be a sensitive biomarker of exposure to polycyclic aromatic hydrocarbons.     

Design and development of cyclohexane-based P,N-ligands for transition metal catalysis

[reaction: see text] A new class of cyclohexane-based P,N-ligands is readily obtained through aziridine ring opening with suitable phosphorus nucleophiles under acidic conditions. trans-1-Amino-2-diphenylphosphinocyclohexane is resolved with tartaric acid to give the final product in >99% ee. The new ligands show high stability toward oxidation at the phosphorus atom.     

Identification and structural characterization by LC‐ESI‐IONTRAP and LC‐ESI‐TOF of some metabolic conjugation products of homovanillic acid in urine of neuroblastoma patients

The levels of urinary catecholamine metabolites, such as homovanillic acid (HVA) and vanillylmandelic acid, are routinely used as a clinical tool in the diagnosis and follow‐up of neuroblastoma (NB) patients. Recently, in the Clinical Pathology Laboratory Unit of G. Gaslini Children Hospital, a commercial method that employs liquid chromatography coupled to electrochemical detection (LC‐EC) has been introduced for the measurement of these metabolites in the routine laboratory practice. Using this LC‐EC method, an unknown peak could be observed only in samples derived from NB patients. To investigate the nature of this peak, we used a combination of liquid chromatography‐time‐of‐flight mass spectrometry (LC‐TOF‐MS) and liquid chromatography‐ion trap tandem mass spectrometry (LC‐IT‐MS). The first approach was used to obtain the elemental composition of the ions present in this new signal. To get additional structural information useful for the elucidation of unknown compounds, the ion trap analyzer was exploited. We were able to identify not just one, but three unknown signals in urine samples from NB patients which corresponded to three conjugated products of HVA: HVA sulfate and two glucuronoconjugate isomers. The enzymatic hydrolysis with β‐glucuronidase confirmed the proposed structures, while the selective alkaline hydrolysis allowed us to distinguish the difference between phenol‐ and acyl‐glucuronide of HVA. The latter was the unknown peak observed in LC‐EC separations of urine samples from NB patients. Copyright © 2012 John Wiley & Sons, Ltd.     

Structures of Solvent-Free,Monomeric LiCCH,NaCCH, and KCCH

The simplest alkali metal acetylides MCCH were made in the gas phase for the first time (see drawing), and their bond lengths were determined by millimeter/submillimeter spectroscopy of their isotopomers. The M−C bond lengths r_CM are the shortest known for organoalkali metal compounds. In the case of LiCCH, the experimentally determined Li−C distance of 1.888 Å has an estimated accuracy of ±0.0005 Å, which should allow a rigorous test of theoretical methods.     

Production of octadienal in the marine diatom Skeletonema costatum

[reaction: see text] Marine diatoms produce alpha,beta,gamma,delta-unsaturated aldehydes that have detrimental effects on the reproduction of their natural predators. The production of these defensive metabolites is suggested to involve enzymatic oxidation of polyunsaturated fatty acids. In this paper, feeding experiments with labeled precursor provide clear evidence in support of the origin of octadienals 1 and 2 from 6,9,12-hexadecatrienoic acid (5), thus proving the involvement of novel lipoxygenase/lyase activity for the oxidation of C(16) fatty acids.     

Exploiting in vitro and in vivo bioluminescence for the implementation of the three Rs principle (replacement,reduction, and refinement) in drug discovery

Bioluminescence-based analytical tools are suitable for high-throughput and high-content screening assays, finding widespread application in several fields related to the drug discovery process. Cell-based bioluminescence assays, because of their peculiar advantages of predictability, possibility of automation, multiplexing, and miniaturization, seem the most appealing tool for the high demands of the early stages of drug screening. Reporter gene technology and the bioluminescence resonance energy transfer principle are widely used, and receptor binding studies of new agonists/antagonists for a variety of human receptors expressed in different cell lines can be performed. Moreover, bioluminescence can be used for in vitro and in vivo real-time monitoring of pathophysiological processes within living cells and small animals. New luciferases and substrates have recently arrived on the market, further expanding the spectrum of applications. A new generation of probes are also emerging that promise to revolutionize the preclinical imaging market. This formidable toolbox is demonstrated to facilitate the implementation of the three Rs principle in the early drug discovery process, in compliance with ethical and responsible research to reduce cost and improve the reliability and predictability of results.     

Multiresidue determination of UV filters in water samples by solid‐phase extraction and liquid chromatography with tandem mass spectrometry analysis

UV filters, contained in sunscreens and other cosmetic products, as well as in some plastics and industrial products, are nowadays considered contaminants of emerging concern because their widespread and increasing use has lead to their presence in the environment. Furthermore, some UV filters are suspected to have endocrine disruption activity. In the present work, we developed an analytical method based on liquid chromatography with tandem mass spectrometry for the determination of UV filters in tap and lake waters. Sixteen UV filters were extracted from water samples by solid‐phase extraction employing graphitized carbon black as adsorbent material. Handling 200 mL of water sample, satisfactory recoveries were obtained for almost all the analytes. The limits of detection and quantification of the method were comparable to those reported in other works, and ranged between 0.7–3.5 and 1.9–11.8 ng/L, respectively; however in our case the number of investigated compounds was larger. The major encountered problem in method development was to identify the background contamination sources and reduce their contribution. UV filters were not detected in tap water samples, whereas the analyses conducted on samples collected from three different lakes showed that the swimming areas are most subject to UV filter contamination.