Indirect ultrasonication for protein quantification and peptide mass mapping through mass spectrometry-based techniques

We report in this work a fast protocol for protein quantification and for peptide mass mapping that rely on ¹⁸O isotopic labeling through the decoupling procedure. It is demonstrated that the purity and source of trypsin do not compromise the labeling degree and efficiency of the decoupled labeling reaction, and that the pH of the labeling reaction is a critical factor to obtain a significant ¹⁸O double labeling. We also show that the same calibration curve can be used for MALDI protein quantification during several days maintaining a reasonable accuracy, thus simplifying the handling of the quantification process. In addition we demonstrate that ¹⁸O isotopic labeling through the decoupling procedure can be successfully used to elaborate peptide mass maps. BSA was successfully quantified using the same calibration curve in different days and plasma from a freshwater fish, Cyprinus carpio, was used to elaborate the peptide mass maps.     

Network Dynamics in Elemental Assimilation and Metabolism

Metabolism and physiology frequently follow non-linear rhythmic patterns which are reflected in concepts of homeostasis and circadian rhythms, yet few biomarkers are studied as dynamical systems. For instance, healthy human development depends on the assimilation and metabolism of essential elements, often accompanied by exposures to non-essential elements which may be toxic. In this study, we applied laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to reconstruct longitudinal exposure profiles of essential and non-essential elements throughout prenatal and early post-natal development. We applied cross-recurrence quantification analysis (CRQA) to characterize dynamics involved in elemental integration, and to construct a graph-theory based analysis of elemental metabolism. Our findings show how exposure to lead, a well-characterized toxicant, perturbs the metabolism of essential elements. In particular, our findings indicate that high levels of lead exposure dysregulate global aspects of metabolic network connectivity. For example, the magnitude of each element’s degree was increased in children exposed to high lead levels. Similarly, high lead exposure yielded discrete effects on specific essential elements, particularly zinc and magnesium, which showed reduced network metrics compared to other elements. In sum, this approach presents a new, systems-based perspective on the dynamics involved in elemental metabolism during critical periods of human development.     

Towards molecular-imprint based SPE of local anaesthetics

Summary Solidago canadensis L., Canadian goldenrod (Asteraceae) has been used in European phytotheraphy for centuries as a component of urological and antiphlogistical remedies. High-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD) and online mass spectrometry (MS) has been used for the separation and quantification of phenolics (chlorogenic acid, caffeic acid, kaempferol-3-O-α-L-rutinoside (nicotiflorin), quercetin-3-O-β-D-rutinoside (rutin), quercetin-3-O-β-D-galactoside (hyperoside), quercetin-3-O-β-D-glucoside (isoquercitrin), quercetin-3-O-β-D-rhamnoside (quercitrin), kaempferol-3-O-α-L-rhamnoside (afzelin) and quercetin from Solidaginis herba. Extracts have been obtained using different technologies. Three aqueous and three alcoholic extracts were studied separately. Reversedphase high-performance liquid chromatography separation of polyphenols on octadecyl sorbent Hypersil was performed, using acetonitrile: acetic acid 2.5 v/v % as eluent in gradient elution. Our results confirm previous reports concerning the presence of several flavonoids. Quantification of the main quercetin glycosides in pharmaceuticals is also reported. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Validated Chiral LC Method for the Enantiomeric Separation of β-Amino-β-(3-Methoxyphenyl) Propionic Acid

A chiral liquid chromatographic method for enantiomeric resolution of β-amino-β-(3-methoxyphenyl) propionic acid was developed and validated. The “hybrid” π-electron donor–acceptor based stationary phase (R,R) Whelk-01 was found to be enantiomerically selective for (R) and (S) enantiomers of β-amino-β-(3-methoxyphenyl) propionic acid with a resolution greater than 2.0. The effects of isopropyl alcohol and ethanol on enantioselectivity and resolution of enantiomers were evaluated. Calibration curves were linear over the range of 0.10–1.00, with a regression coefficient (r) of 0.999. The limit of detection (LOD) and limit of quantification (LOQ) were 300 and 1,000 ng mL⁻¹ respectively for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of (S) enantiomer at LOQ concentration was 2.8. The percentage recovery of (S) enantiomer from (R) enantiomer samples ranged from 92 to 102. The test solution was observed to be stable up to 24 h after the preparation. The developed normal phase chiral LC method can be used for the enantiomeric purity evaluation of R-β-amino-β-(3-methoxyphenyl) propionic acid.     

Sample cleanup-free determination of mycophenolic acid and its glucuronide in serum and plasma using the novel technology of ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry

Mycophenolic acid (MPA) is an immunosuppressant drug which powerfully inhibits lymphocyte proliferation. Since the early 1990s it has been used to prevent rejection in organ transplantation. The requirement of therapeutic drug monitoring shown in previous studies raises the necessity of acquiring accurate and sensitive methods to measure MPA and its major metabolite mycophenolic acid glucuronide (MPAG).The authors developed a sample cleanup-free, rapid, and highly specific method for simultaneous measurement of MPA and MPAG in human plasma and serum using the novel technology of ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry. MPA- and MPAG-determinations were performed during a 2.0-min run time. Multiple calibration curves for the analysis of MPA and MPAG exhibited consistent linearity and reproducibility in the range of 0.05-100 (r > 0.999) mg L⁻¹ and 4-4000 mg L⁻¹ (r > 0.999), respectively. Limits of Detection were 0.014 mg L⁻¹ for MPA and 1.85 mg L⁻¹ for MPAG. Lower Limits of Quantification were 0.05 mg L⁻¹ for MPA and 2.30 mg L⁻¹ for MPAG. Interassay imprecision was <10% for both substances. Mean recovery was 103.6% (range 78.1-129.7%) for MPA and 111.1% (range 73.0-139.6%) for MPAG. Agreement was good for MPA and MPAG between the presented method and a validated HPLC-MS/MS method. The Passing-Bablok regression line for MPA and MPAG was HPLC-MS/MS = 1.14 UPLC-MS/MS—0.14 [mg L⁻¹], r = 0.96, and HPLC-MS/MS = 0.77 UPLC-MS/MS + 0.50 [mg L⁻¹], r = 0.97, respectively. This sample cleanup-free and robust LC-MS/MS assay facilitates the rapid, accurate and simultaneous determination of MPA and MPAG in human body fluids.     

A Validated Chiral LC Method for the Determination of Enantiomeric Purity of Pemetrexed Disodium on an Amylose-Based Chiral Stationary Phase

A simple and new isocratic normal phase chiral HPLC method has been developed for the determination of enantiomeric purity of pemetrexed disodium (l-enantiomer) in bulk drugs with a short run time of about 20 min. Chromatographic separation of l and d-enantiomers of pemetrexed disodium was achieved on an amylose based chiral stationary phase using a mobile phase consists of hexane, ethanol and trifluoro acetic acid. The resolution between the enantiomers was found to be more than 2.0. The system precision and method precision were found to be within 5% RSD for the distomer (d-enantiomer) at its specification level (i.e. not more than 1.0% w/w). The limit of detection and limit of quantification of distomer were 1.6 and 5 μg mL⁻¹, respectively for 10 μL injection volume. The percentage recovery of distomer was ranged from 90.6 to 105.7 in bulk drug samples. The test solution was found to be stable in the diluent for 48 h. The method was found to be specific for the enantiomers of pemetrexed disodium and can be conveniently used for the quantification of undesired d-enantiomer present in the bulk drug samples of pemetrexed disodium.     

Grid method: Application to the characterization of cracks

In this paper we present an application of the grid method to the assessment of crack initiation and growth in brittle materials. It is shown that a grid deposited on the surface of a structure can be utilized to characterize the crack location and opening. Furthermore, one single picture only is required to carry out a control over a predefined area. First, the principle of the method is introduced, showing that a periodical pattern encoding a cracked surface can be analyzed by signal processing. The phase modulation of the grid can be calculated very accurately for any small deformation of the structure. Discontinuities due to cracks are easily detected on the phase field and the crack width is proportional to the amplitude of the discontinuity. Secondly, the performances of the method are presented, mainly depending on the spatial frequency of both the pattern and the sampling. It is shown that good results can generally be obtained with few setting difficulties and equipment investment. Finally, we sum up some basic recommendations for the practical use of the technique.     

Highly sensitive non‐isotopic restriction endonuclease fingerprinting of nucleotide variability in the gp60 gene within Cryptosporidium species,genotypes and subgenotypes infective to humans,and its implications

The high‐resolution analysis of genetic variation has major implications for the identification of parasites and micro‐organisms to species and subspecies as well as for population genetic and epidemiological studies. In this study, we critically assessed the effectiveness of a PCR‐based restriction endonuclease fingerprinting (REF) method for the detection of mutations in the 60 kDa glycoprotein gene (gp60) of Cryptosporidium, a genus of parasitic protists of major human and animal health importance globally. This gene displays substantial intraspecific variability in sequence, particularly in a TCA (perfect and imperfect) microsatellite region, is present as a single copy in the nuclear genome and is used widely as a marker in molecular epidemiological studies of Cryptosporidium hominis and C. parvum, the two predominant species that infect humans. The results of this study demonstrated an exquisite capacity of REF to detect nucleotide variability in the gp60 gene within each of the two species. The differentiation of genotypes/subgenotypes based on REF analysis was supported by targeted sequencing, allowing the detection of levels of variation as low as a single‐nucleotide transversion for amplicons of ∼1 kb in size. The high‐throughput potential and relatively low‐cost of REF make it a particularly useful tool for large‐scale genetic analyses of C. hominis and C. parvum. REF could also be utilized for comparative surveys of genetic variability across large nuclear genomic regions. Such analyses of Cryptosporidium in clinical and environmental samples by REF have important implications for identifying sources of infection, modes of transmission and/or possible infectivity to humans, thus assisting in the surveillance and control of cryptosporidiosis. Given its excellent mutation detection capacity, REF should find broad applicability to various single‐copy genes as well as a wide range of other protozoan and metazoan parasites. (The nucleotide sequences reported in this article are available in the GenBank database under accession numbers GU214343–GU214371).     

On realizations of point determining graphs, and obstructions to full homomorphisms

A graph is point determining if distinct vertices have distinct neighbourhoods. A realization of a point determining graph H is a point determining graph G such that each vertex-removed subgraph G-x which is point determining, is isomorphic to H. We study the fine structure of point determining graphs, and conclude that every point determining graph has at most two realizations.A full homomorphism of a graph G to a graph H is a vertex mapping f such that for distinct vertices u and v of G, we have uv an edge of G if and only if f(u)f(v) is an edge of H. For a fixed graph H, a full H-colouring of G is a full homomorphism of G to H. A minimal H-obstruction is a graph G which does not admit a full H-colouring, such that each proper induced subgraph of G admits a full H-colouring. We analyse minimal H-obstructions using our results on point determining graphs. We connect the two problems by proving that if H has k vertices, then a graph with k+1 vertices is a minimal H-obstruction if and only if it is a realization of H. We conclude that every minimal H-obstruction has at most k+1 vertices, and there are at most two minimal H-obstructions with k+1 vertices.We also consider full homomorphisms to graphs H in which loops are allowed. If H has ? loops and k vertices without loops, then every minimal H-obstruction has at most (k+1)(?+1) vertices, and, when both k and ? are positive, there is at most one minimal H-obstruction with (k+1)(?+1) vertices.In particular, this yields a finite forbidden subgraph characterization of full H-colourability, for any graph H with loops allowed.     

Quantification of testosterone undecanoate in human hair by liquid chromatography–tandem mass spectrometry

Testosterone undecanoate (T‐C11) can be used by athletes in order to improve performance. After oral intake, T‐C11 is rapidly metabolized, hampering discrimination between exogenous and endogenous testosterone. A possible alternative is to detect the intact ester in hair. A method based on liquid chromatography–tandem mass spectrometry was developed for the determination of T‐C11 in hair. The sample procedure consisted of digestion of 200 mg of pulverized hair with tris(2‐carboxyethyl)phosphine hydrochloride and liquid–liquid extraction with n‐pentane. Several parameters such as the mobile phase, the ionization source and the washing step were optimized. The method was validated at different spiked levels obtaining satisfactory values for accuracy (between 92 and 102%) with relative standard deviations lower than 7% and a limit of detection of 0.2 ng/g. The applicability of the method was checked by the analysis of three samples from patients using T‐C11. A peak for the analyte was detected in all samples with concentrations between 0.4 and 8.4 ng/g. Copyright © 2009 John Wiley & Sons, Ltd.