Multiplexed immunoassay to detect anabolic androgenic steroids in human serum

A multianalyte enzyme-linked immunosorbent assay (ELISA) has been developed for the simultaneous detection of anabolic androgenic steroids (AAS) in human serum. The multiplexed method was developed according to a planar strategy in which the analytes are identified by their location in the microtiter plate. In the immunochemical procedure established here, human serum samples are mixed with a cocktail of antibodies and added to the distinct sections of a microplate biofunctionalized with different haptenized biomolecules. The cocktail of antibodies consists of a mixture of polyclonal antibodies raised against stanozolol (ST), boldenone (B), and tetrahydrogestrinone (THG). The whole immunochemical analytical procedure takes around 2 h including sample preparation, and many samples can be processed simultaneously to screen for the presence of the three AAS in a single run. Using this ELISA, ST, B, and THG can be detected and quantified individually. When used as a screening method, due to the cross-reactivity profiles of the immunoreagents used, the presence of up to 11 AAS can be detected simultaneously. The detectabilities achieved by this method in human serum are below the MRPLs (minimum required performance limits) proposed by WADA (World Anti-Doping Agency) and reference laboratories of the European Community.     

Development of a simultaneous analytical method for selected anorectics,methamphetamine, MDMA,and their metabolites in hair using LC-MS/MS to prove anorectics abuse

Owing to the tight control of methamphetamine, it is presumed that phentermine, an amphetamine-type anorectic, has recently been considered a supplement for methamphetamine abusers in Korea. In addition, the abuse of other anorectics obtained by inappropriate means has become a social issue. Hair is a useful specimen to prove chronic drug use. Therefore, an analytical method for the simultaneous detection of phentermine, phendimetrazine, amfepramone, fenfluramine, mazindol, methamphetamine, and 3,4-methylenedioxymethamphetamine (MDMA), as well as their metabolites, which covers the major amphetamines and anorectic agents in Korea, in hair was established and validated using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The drugs and their metabolites in hair were extracted using 1 % HCl in methanol and then filtered and analyzed by LC-MS/MS with electrospray ionization in positive mode. The validation results for selectivity, linearity, matrix effect, recovery, process efficiency, intra- and interassay precision and accuracy, and processed sample stability were satisfactory. The limits of detection ranged from 0.025 to 1 ng/10 mg hair and the limits of quantification were 0.25 ng/10 mg hair for every analyte except mazindol and phentermine, for which they were 10 ng/10 mg hair. The method was successfully applied for the segmental determination of selected anorectics, methamphetamine, MDMA, and their metabolites in hair from 39 drug suspects. Among the anorectics, phentermine and/or phendimetrazine were identified with or without methamphetamine in the hair samples. Closer supervision of the inappropriate use of anorectics is necessary. Also, hair analysis is useful for monitoring the abuse potential of unnoticed drugs.     

Synthesis and stereochemistry of some novel dihydropyrrolo[3,4-c]pyrazoles

Abstract  

Eleven novel dihydropyrrolo[3,4-c]pyrazole derivatives were obtained by the reaction of chiral (1R)-N-(1-phenylethyl)maleimide and C,N-aryl-substituted nitrilimines. The reaction afforded the cycloadducts as a regioisomeric mixture which can be separable in some cases. The structure and stereochemistry of cycloadducts were assigned on the basis of infrared (IR), ¹H and ¹³C nuclear magnetic resonance (NMR), mass and X-ray spectra, optical rotation measurements, and CHN analyses.     

Crystal habit modifications of imatinib mesylate under various precipitation conditions

Abstract  

Crystals of the α-form of imatinib mesylate with various habits (e.g., polyhedral-like and plate-like) were prepared from various organic solvents (e.g., butyl lactate, 4-methyl-2-pentanone, 2-methyl-2-butanol, 2-isopropoxyethanol, propyl ether) by several precipitation methods. The methods provide imatinib mesylate in a non-needle-shaped crystalline α-form. The crystal modification was identified by hot-stage microscopy, scanning electron microscopy, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and X-ray powder diffraction (XRPD). The analyses by DSC, IR, and XRPD indicate that imatinib mesylate crystals with various habits have the same crystal structure. The plate-like habit has been also observed in the system where the organic solvent acts as a precipitant.     

What determines MALDI ion yields? A molecular dynamics study of ion loss mechanisms

Ion recombination in matrix-assisted laser desorption/ionization (MALDI) is as important as any ion formation process in determining the quantity of ions observed but has received comparatively little attention. Molecular dynamics simulations are used here to investigate some models for recombination, including a Langevin-type model, a soft threshold model and a tunneling model. The latter was found to be superior due to its foundations in a widespread physical phenomenon, and its lack of excessive sensitivity to parameter choice. Tunneling recombination in the Marcus inverted region may be a major reason why MALDI is a viable analytical method, by allowing ion formation to exceed ion loss on the time scale of the plume expansion. Ion velocities, photoacoustic transients and pump-probe measurements might be used to investigate the role of recombination in different MALDI matrices, and to select new matrices.     

Membrane-assisted solvent extraction coupled to large volume injection–gas chromatography–mass spectrometry for the determination of a variety of endocrine disrupting compounds in environmental water samples

Membrane-assisted solvent extraction coupled to large volume injection in a programmable temperature vaporisation injector using gas chromatography–mass spectrometry analysis was optimised for the simultaneous determination of a variety of endocrine disrupting compounds in environmental water samples (estuarine, river and wastewater). Among the analytes studied, certain hormones, alkylphenols and bisphenol A were included. The nature of membranes, extraction solvent, extraction temperature, solvent volume, extraction time, ionic strength and methanol addition were evaluated during the optimisation of the extraction. Matrix effects during the extraction step were studied in different environmental water samples: estuarine water, river water and wastewater (influent and effluent). Strong matrix effects were observed for most of the compounds in influent and effluent samples. Different approaches were studied in order to correct or minimise matrix effects, which included the use of deuterated analogues, matrix-matched calibration, standard addition calibration, dilution of the sample and clean-up of the extract using solid-phase extraction (SPE). The use of deuterated analogues corrected satisfactorily matrix effect for estuarine and effluent samples for most of the compounds. However, in the case of influent samples, standard addition calibration and dilution of the sample were the best approaches. The SPE clean-up provided similar recoveries to those obtained after correction with the corresponding deuterated analogue but better chromatographic signal was obtained in the case of effluent samples. Method detection limits in the 5–54 ng L⁻¹ range and precision, calculated as relative standard deviation, in the 2–25% range were obtained.     

Bioaccessibility of total arsenic and arsenic species in seafood as determined by a continuous online leaching method

A continuous leaching method coupled online with inductively coupled plasma mass spectrometry (ICP-MS) detection was used to assess the maximum bioaccessibility of arsenic (As) in seafood samples. The method simulates continuous-flow digestion by successively pumping artificial saliva, gastric and intestinal juices through a mini-column of powdered sample directly connected to the nebuliser of an ICP-MS instrument. The method allows the real-time measurement of As being released by a given reagent. Because the analyte is continuously removed from the system, in contrast to batch methods, the dissolution equilibrium is driven to the right, hence quickly providing information about the worst-case scenario. Following consecutive leaching by the digestive reagents, the leachates were subject to speciation analysis by ion-exchange chromatography with ICP-MS detection to determine the arsenic species released. Finally, the remaining residue from the mini-column was fully digested to verify mass balance. The method was used to determine the bioaccessibility of total As and As species in four certified reference materials and in several real seafood samples. The mass balance was verified in each case. Generally speaking, the non-toxic form was easily released whereas the inorganic forms were poorly bioaccessible.     

A lectin-coupled,multiple reaction monitoring based quantitative analysis of human plasma glycoproteins by mass spectrometry

Aberrant protein glycosylation may be closely associated with cancer pathology. To measure the abundance of protein glycoforms with a specific glycan structure in plasma samples, we developed a lectin-coupled multiple reaction monitoring (MRM)-based mass spectrometric method. It was confirmed that the method could provide reproducible results with precision sufficient to distinguish differences in the abundance of protein glycoforms between individuals. Plasma samples prepared from hepatocellular carcinoma (HCC) patients without immuno-depletion of highly abundant plasma proteins were fractionated by use of fucose-specific aleuria aurantia lectin (AAL) immobilized on magnetic beads by use of a biotin–streptavidin conjugate. The lectin-captured fractions were digested by trypsin and profiled by tandem mass spectrometry. From the proteomic profiling data, target glycoproteins were selected and analyzed quantitatively by MRM-based analysis. The reproducibility of MRM-based quantification of the selected target proteins was reliable, with precision (CV; ≤14% for batch-to-batch replicates and ≤19% for replicates over three days) sufficient to distinguish differences in the abundance of AAL-captured glycoforms between individual plasma samples. This lectin-coupled, MRM-based method, measuring only lectin-captured glycoforms of a target protein rather than total target protein, is a tool for monitoring differences between individuals by measuring the abundance of aberrant glycoforms of a target protein related to a disease. This method may be further applied to rapid verification of biomarker candidates involved in aberrant protein glycosylation in human plasma.     

Determination of lipophilicity for antitumor acridinone derivatives supported by gradient high-performance liquid chromatography method

The lipophilicity values of selected acridinone (imidazoacridinone and triazoloacridinone) derivatives were measured by gradient reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 stationary phase with a water/acetonitrile mixture as a mobile phase. The retention times obtained served as input data and appropriate log k_w values (i.e., the retention factor log k_w extrapolated to 0% organic modifier) as an alternative to log P were calculated using the DryLab program. The relationships between the lipophilicity (log k_w) and the chemical structure of the studied compounds, as well as correlation between experimentally determined lipophilicities (log k_w) and log P data calculated using some commonly available software, are discussed.     

Fast counter-electroosmotic capillary electrophoresis–time-of-flight mass spectrometry of hyaluronan oligosaccharides

Fast capillary electrophoresis–mass spectrometry measurements under counter-electroosmotic analyte migration conditions are presented. Efficient separations of a homologous series of six hyaluronan oligosaccharides (comprising 1–6 hyalobiuronic acid moieties) could be completed in 65 s. Separations were achieved in short-length fused silica capillaries under high electric field strengths of up to 1.25 kV·cm⁻¹. Capillary inner diameters ranging from 5 to 50 μm were investigated, resulting in an optimal value of 15 μm. The influence of capillary dimensions and buffer composition on separation efficiency and sensitivity are discussed. Optimal separations were achieved using a 28 cm × 15 μm capillary, a separation high voltage of 35 kV, a background electrolyte of 25 mM ammonium acetate adjusted to pH 8.5, and negative ionization mode. The optimized method was successfully applied to a bovine testicular hyaluronidase digest of hyaluronan. Only minimal sample pretreatment for protein-containing samples is required. The simple manual injection procedure and fast separations allow for a sample throughput of 35 samples per hour.