HPLC assay for determination of amphotericin B in biological samples

A fast and selective HPLC method for assaying amphotericin B in biological samples was developed and validated. The chromatographic separation was achieved in less than 12 min on a reverse-phase C(18) column using an acetonitrile-acetic acid-water (52:4.3:43.7, v/v/v) mixture as mobile phase. The flow rate was 1 mL/min and the effluent was monitored at 406 nm. A linear response over the concentration range 0.1-10.0 microg/mL was obtained. Intra-day and inter-day RSDs were below 5% for all the sample types. This new HPLC method was applied to assay amphotericin B in plasma and several tissue samples such as kidney, liver, spleen and bone marrow. Application of this method to pharmacokinetic studies in mice and dog is provided.     

Enzymatic mechanisms of biological magnetic sensitivity: nuclear spin effects

Intracellular enzymatic reactions involving ion-radical states are shown to act as a universal mechanism of magnetically sensitive living organisms. Weak magnetic fields can affect the rate of intracellular enzymatic reactions. The main magnetically sensitive stage is the singlet-triplet conversion of ion-radical pairs in the active sites of enzymes induced by Zeeman and hyperfine interactions of electron and nuclear spins. The participation of a nuclear spin in the ion-radical process results in a strong dependence of the enzymatic reaction rate in weak magnetic fields comparable with the magnetic field of the Earth.     

A review of chromatographic methods for the assessment of phospholipids in biological samples

Phospholipids are important constituents of all living cell membranes. Lipidomics is a rapidly growing field that provides insight as to how specific phospholipids play roles in normal physiological and disease states. There are many analytical methods available for the qualitative and quantitative determination of phospholipids. This review provides a summary of the methods that were historically used such as thin layer chromatography, gas chromatography and high-performance liquid chromatography. In addition, an introduction to applications of interfacing these traditional chromatographic techniques with mass spectrometry is provided.     

Characterization and classification of matrix effects in biological samples analyses

An exhaustive classification of matrix effects occurring when a sample preparation is performed prior to liquid-chromatography coupled to mass spectrometry (LC–MS) analyses was proposed. A total of eight different situations were identified allowing the recognition of the matrix effect typology via the calculation of four recovery values. A set of 198 compounds was used to evaluate matrix effects after solid phase extraction (SPE) from plasma or urine samples prior to LC–ESI-MS analysis. Matrix effect identification was achieved for all compounds and classified through an organization chart. Only 17% of the tested compounds did not present significant matrix effects.     

Conformational changes of hapten-protein conjugates resulting in improved broad-specificity and sensitivity of an ELISA for organophosphorus pesticides

The type of hapten linkage to a carrier protein can play an important role in determining the nature of the resulting antibody response. Generic haptens using three types of linkers were synthesized (a monocarboxylic acid, an unsaturated hydrocarbon and a carboxamido spacer). These haptens were conjugated to bovine serum albumin (BSA) and used as immunogens to produce broad-specificity monoclonal antibodies (MAbs) to organophosphorus pesticides (OPs). Three-dimensional (3D) structures of hapten-lysine conjugates were optimized using molecular modeling (MM) to mimic conformations of hapten-BSA conjugates. The results from MM studies revealed a change of the 3D conformation and electrostatic potential of hapten 1 when the monocarboxylic acid linker was coupled to lysine. This result was consistent with the observed high-cross-reactivity of the corresponding MAb-H1 for the OPs. The competitive indirect enzyme-linked immunosorbent assay based on MAb-H1 is ideally suited to be used as a screening method for OP contaminants.     

Preliminary study of the effects of some physical parameters on the stability of methylmercury in biological samples.

The effects of storage conditions (long-term storage of wet samples in a deep-freeze or thermal cycling), freeze-drying and gamma-irradiation at 1 and 5 Mrad on the stability of methylmercury in some biological samples were investigated. Methylmercury was determined by volatilization separation followed by gas chromatography and by ion-exchange separation of inorganic and organic species followed by measurement by cold vapour atomic absorption spectrometry (CVAAS). Total mercury was determined by CVAAS. Biological samples studied included fish and shellfish tissues, human hair and blood samples and appropriate reference materials. From the preliminary results obtained it can be concluded that fresh and dried fish muscle and fish certified reference materials show good stability with time and against temperature cycling. Shellfish and blood should not be repeatedly frozen and unfrozen otherwise possible losses of methylmercury can occur. Losses of methylmercury of up to 30% from wet mussels occurred on prolonged storage in a deep-freeze. Gamma-irradiation reduced the methylmercury content of the fish and shellfish only for hake (Merluccius merluccius). Further experiments should be carried out to confirm this and to investigate if this effect is species dependent. Apparent losses of methylmercury on freeze-drying of blood need to be reconfirmed on further samples.     

Optimizing methylation conditions for gas liquid chromatography assay of lactic and succinic acid in biological samples

Summary Aqueous standard solutions of lactic and succinic acids were methylated for assay by gas-liquid chromatography. The efficiency of methylation using boron trifluoridemethanol reagent was compared to methylation using a sulfuric acid-methanol mixture; the ratio of sulfuric acid and methanol was also varied. Reactions were done at 100°C/5 min and at room temperature/20 hours. We found the sulfuric acid-methanol was most efficient for a rapid assay; however, the ratio of sulfuric acid and methanol used is critical in the assay of lactic acid and would be especially important in the clinical laboratory using gas-liquid chromatography for analysis of cerebrospinal fluid from suspect meningitis patients or other body fluids.     

Liquid chromatography with pre-column dansyl derivatisation and fluorimetric detection applied to the assay of morphine in biological samples

A simple method employing pre-column dansylation and liquid chromatography is proposed for a very sensitive and specific assay of morphine in biological samples. Nalorphine is used as an internal standard. The detection limit is 0.2 picomol of injected morphine. In the assay of human sera spiked with 150 nmol/l, the intra- and inter-assay coefficients of variation were 3.7% (n = 10) and 4.5% (n = 10), respectively. No interferences were observed from more than 70 opiate and non-opiate drugs. Urine, plasma and total blood were assayed, using different extraction methods, with negligible interference from coextractives.     

Validated capillary electrophoresis assay for the simultaneous enantioselective determination of propafenone and its major metabolites in biological samples

A robust, inexpensive, and fully validated CE method for the simultaneous determination of the enantiomers of propafenone (PPF), 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF) in serum and in in vitro media is described. It is based upon liquid-liquid extraction at alkaline pH followed by analysis of the reconstituted extract by CE in presence of a pH 2.0 running buffer composed of 100 mM sodium phosphate, 19% methanol, and 0.6% highly sulfated beta-CD. For each compound, the S-enantiomers are shown to migrate ahead of their antipodes, and the overall run time is about 30 min. Enantiomer levels between 25 and 1000 ng/mL provide linear calibration graphs, and the LOD for all enantiomers is between 10 and 12 ng/mL. The assay is shown to be suitable for the determination of the enantiomers of PPF and its metabolites in in vitro incubations comprising human liver microsomes or single CYP450 enzymes (SUPERSOMES). Incubations with CYP2D6 SUPERSOMES revealed, for the first time, the simultaneous formation of the enantiomers of 5OH-PPF and NOR-PPF with that enzyme. CE data can be used for the evaluation of the enzymatic N-dealkylation and hydroxylation rates.     

Validity of extracellular water assessment with saliva samples using plasma as the reference biological fluid

Extracellular water (ECW) assessment is based on dilution techniques, commonly using blood sampling. However, plasma collection is an invasive procedure. We aimed to validate the use of saliva for ECW estimation by the bromide dilution technique using plasma as the reference method, in a sample of elite athletes. A total of 89 elite athletes with a mean age of 20.4 ± 4.4 years were evaluated. Baseline samples were collected before sodium bromide oral dose administration, and enriched samples were collected 3 h post‐dose administration. The bromide concentration was assessed by high‐performance liquid chromatography. Comparison of means, concordance coefficient correlation (CCC), multiple regression and Bland–Altman analysis were performed. The ECW from saliva explained 91% of the variance in ECW by plasma with a standard error of estimation of 0.91 kg. The CCC between alternative and reference methods was 0.952. No significant trend was observed between the mean and difference of the methods, with limits of agreement ranging between ?1.5 and 2.1 kg. These findings reveal that bromide dilution volume calculated from saliva samples is a valid noninvasive method for ECW assessment in elite athletes. Copyright © 2012 John Wiley & Sons, Ltd.     

Hapten synthesis and enzyme-linked immunosorbent assay for phosmet residues: assay optimization and investigation of matrix effects from different food samples

In this study, a panel of haptens was synthesized for immunoconjugate preparation, and several haptens for heterologous tracer conjugates were also prepared. A highly sensitive polyclonal antibody against the organophosphorus insecticide phosmet was obtained and competitive direct enzyme-linked immunosorbent assays (cd-ELISA) for this pesticide were developed. In the cd-ELISA, the limit of detection (IC₁₅) was 0.6 μg kg⁻¹ and the sensitivity (IC₅₀) was 20 μg kg⁻¹. The suitability of the ELISA for pesticide quantification in peach, apple, orange juice, and apple juice was also studied. Good accuracy and precision were obtained with mean recoveries between 78% and 102.3% and mean coefficients of variation below 13.63%. Validation of the ELISA was conducted by high-performance liquid chromatography. The correlation between the data obtained using the microwell assay and the high-performance liquid chromatography was good (R ² = 0.9849). The developed immunoassay methods were suitable for the rapid quantitative or qualitative determination of phosmet in food samples.     

Determination of thallium in biological samples

Determination of thallium has become a major interest because of its high toxicity, especially as the monovalent cation. Thallium poisoning in the human body must be checked quickly by analysis of biological samples. This review highlights the development of highly sensitive detection techniques applied to the determination of thallium in biological samples, with or without pretreatment, based on the literature compiled in Analytical Abstracts from 1990.     

Isolation of thorium in biological samples

A scheme for the isolation of subpicogram to microgram amounts of thorium from bone and soft tissue samples is described. Thorium is first collected by co-precipitation with iron(III) hydroxide and then adsorbed by a cation-exchange resin column from a concentrated hydrochloric acid solution of the precipitate. A double precipitation is necessary for bone samples whereas a single precipitation suffices for liver samples. The thorium is eluted from the column with an ammonium carbonate solution which is then evaporated to dryness to effect the isolation. Good recoveries of thorium from bone samples are obtained.