Confirmatory method for the determination of various acetylgestagens in animal kidney fat using liquid chromatography-tandem mass spectrometry

A confirmatory method has been developed and validated that allows for the simultaneous detection of medroxyprogesterone acetate (MPA), megestrol acetate (MGA), melengestrol acetate (MLA), chlormadinone acetate (CMA) and delmadinone acetate (DMA) in animal kidney fat using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The compounds were extracted from kidney fat using acetonitrile, defatted using a hexane wash and subsequent saponification. Extracts were then purified on Isolute CN solid-phase extraction cartridges and analysed by LC-MS/MS. The method was validated in animal kidney fat in accordance with the criteria defined in Commission Decision 2002/657/EC. The decision limit (CC) was calculated to be 0.12, 0.48, 0.40, 0.63 and 0.54 g kg-1, respectively, for MPA, MGA, MLA, DMA and CMA, with respective detection capability (CC) values of 0.20, 0.81, 0.68, 1.07 and 0.92 g kg-1. The measurement uncertainty of the method was estimated at 16, 16, 19, 27 and 26% for MPA, MGA, MLA, DMA and CMA, respectively. Fortifying kidney fat samples (n = 18) in three separate assays showed the accuracy of the method to be between 98 and 100%. The precision of the method, expressed as % RSD, for within-laboratory reproducibility at three levels of fortification (1, 1.5 and 2 g kg-1 for MPA, 5, 7.5 and 10 g kg-1 for MGA, MLA, DMA and CMA) was less than 5% for all analytes.

Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 mug kg (-1) in liver and 5, 15 and 50 mug kg (-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 mug kg (-1), respectively. (C) 2003 Elsevier Science B.V. All rights reserved.

DETECTION OF CLENBUTEROL RESIDUES IN BOVINE LIVER, MUSCLE, RETINA AND URINE USING GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

A gas chromatographic/mass spectrometric method is described for the detection of clenbuterol residues in liver, muscle, urine and retina. Tissue samples are first digested using protease and any clenbuterol present is extracted using a simple liquid/liquid extraction procedure. The dried extracts are then derivatized using methylboronic acid and the derivatives are subjected to gas chromatography/mass spectrometry on a magnetic sector instrument. The detection limit of the assay is 0.05 ng g-1 clenbuterol in liver, muscle or urine using a 10 g sample size, and 4 ng g-1 in retina using a 0.5 g sample size. The assay is made very specific by using selected ion monitoring of three ions at a resolution of 3500 and by ion ratio measurements. The precision and reproducibility of the assay are enhanced by the use of a deuterated internal standard, with a typical coefficient of variation of 3%.

Femtosecond lasers for mass spectrometry: Proposed application to catalytic hydrogenation of butadiene

Mass spectra from the interaction of intense, femtosecond laser pulses with 1,3-butadiene, 1-butene, and n-butane have been obtained. The proportion of the fragment ions produced as a function of intensity, pulse length, and wavelength was investigated. Potential mass spectrometry applications, for example in the analysis of catalytic reaction products, are discussed.

Validation of membrane protein topology models by oxidative labeling and mass spectrometry

Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ·OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424). Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ·OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

Profiling excretory/secretory proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry

Infection of mammalian skeletal muscle with the intracellular parasite Trichinella spiralis results in profound alterations in the host cell and a realignment of host cell gene expression. The role of parasite excretory/secretory (E/S) products in mediating these effects is unknown, largely due to the difficulty in identifying and assigning function to individual proteins. In this study, we have used two-dimensional electrophoresis to analyse the profile of muscle larva excreted/secreted proteins and have coupled this to protein identification using MALDI-TOF mass spectrometry. Interpretation of the peptide mass fingerprint data has relied primarily on the interrogation of a custom-made Trichinella EST database and the NemaGene cluster database for T. spiralis. Our results suggest that this proteomic approach is a useful tool to study protein expression in Trichinella spp. and will contribute to the identification of excreted/secreted proteins.

Can we trust mass spectrometry for determination of arsenic peptides in plants:comparison of LC-ICP-MS and LC-ES-MS/ICP-MS with XANES/EXAFS in analysis of Thunbergia alata

The weakest step in the analytical procedure for speciation analysis is extraction from a biological material into an aqueous solution which undergoes HPLC separation and then simultaneous online detection by elemental and molecular mass spectrometry (ICP-MS/ES-MS). This paper describes a study to determine the speciation of arsenic and, in particular, the arsenite phytochelatin complexes in the root from an ornamental garden plant Thunbergia alata exposed to 1 mg As L(-1) as arsenate. The approach of formic acid extraction followed by HPLC-ES-MS/ICP-MS identified different As(III)-PC complexes in the extract of this plant and made their quantification via sulfur (m/z 32) and arsenic (m/z 75) possible. Although sulfur sensitivity could be significantly increased when xenon was used as collision gas in ICP-qMS, or when HR-ICP-MS was used in medium resolution, the As:S ratio gave misleading results in the identification of As(III)-PC complexes due to the relatively low resolution of the chromatography system in relation to the variety of As-peptides in plants. Hence only the parallel use of ES-MS/ICP-MS was able to prove the occurrence of such arsenite phytochelatin complexes. Between 55 and 64% of the arsenic was bound to the sulfur of peptides mainly as As(III)(PC(2))(2), As(III)(PC(3)) and As(III)(PC(4)). XANES (X-ray absorption near-edge spectroscopy) measurement, using the freshly exposed plant root directly, confirmed that most of the arsenic is trivalent and binds to S of peptides (53% As-S) while 38% occurred as arsenite and only 9% unchanged as arsenate. EXAFS data confirmed that As-S and As-O bonds occur in the plants. This study confirms, for the first time, that As-peptides can be extracted by formic acid and chromatographically separated on a reversed-phase column without significant decomposition or de-novo synthesis during the extraction step.

The Use of Electrospray Mass Spectrometry to Determine Speciation in a Dynamic Combinatorial Library for Anion Recognition

The composition of a dynamic mixture of similar 2,2'-bipyridine complexes of iron(II) bearing either an amide (5-benzylamido-2,2'-bipyridine and 5-(2-methoxyethane)amido-2,2'-bipyridine) or an ester (2,2'-bipyridine-5-carboxylic acid benzylester and 2,2'-bipyridine-5-carboxylic acid 2-methoxyethane ester) side chain have been evaluated by electrospray mass spectroscopy in acetonitrile. The time taken for the complexes to come to equilibrium appears to be dependent on the counteranion, with chloride causing a rapid redistribution of two preformed heteroleptic complexes (of the order of 1 hour), whereas the time it takes in the presence of tetrafluoroborate salts is in excess of 24^^h. Similarly the final distribution of products is dependent on the anion present, with the presence of chloride, and to a lesser extent bromide, preferring three amide-functionalized ligands, and a slight preference for an appended benzyl over a methoxyethyl group. Furthermore, for the first time, this study shows that the distribution of a dynamic library of metal complexes monitored by ESI-MS can adapt following the introduction of a different anion, in this case tetrabutylammonium chloride to give the most favoured heteroleptic complex despite the increasing ionic strength of the solution. 

Maximum Residue Level validation of triclabendazole marker residues in bovine liver, muscle and milk by UHPLC tandem mass spectrometry.

Triclabendazole is the only anthelmintic drug, which is active against immature, mature and adult stages of fluke. The objective of this work was to develop an analytical method to quantify and confirm the presence of triclabendazole residues around the MRL. In this work, a new analytical method was developed, which extended dynamic range to 1–100 and 5–1000 g kg-1 for milk and tissue, respectively. This was achieved using a mobile phase containing trifluoroacetic acid (pKa of 0.3), which resulted in the formation of the protonated pseudomolecular ions, [M+H]+, of triclabendazole metabolites. Insufficient
ionisation of common mobile phase additives due to low pKa values (<2) was identified as the cause of poor linearity. The new mobile phase conditions allowed the analysis of triclabendazole residues in liver, muscle and milk encompassing their EU maximum residue levels (MRL) (250, 225 and 10 g kg-1 respectively). Triclabendazole residues were extracted using a modified QuEChERS method and analysed by positive electrospray ionisation mass spectrometry with all analytes eluted by 2.23 min. The method was validated at the MRL according to Commission Decision (CD) 2002/657/EC criteria. The decision limit (CC) of the method was in the range of 250.8–287.2, 2554.9–290.8 and 10.9–12.1 g kg-1 for liver, muscle and milk, respectively. The performance of the method was successfully verified for triclabendazole in muscle by participating in a proficiency study, the method was also applied to incurred liver, muscle and milk samples.

Human aflatoxin albumin adducts quantitatively compared by ELISA, HPLC with fluorescence detection, and HPLC with isotope dilution mass spectrometry

Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B-1. In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r(2) = 0.95) and 3.3 (r(2) = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen.