Neutral loss and precursor ion scan tandem mass spectrometry for study of activated benzopyrene-DNA adducts.

Methodology for detection of activated benzo[a]pyrene (B[a]P)-nucleoside adducts by liquid chromatography-tandem mass spectrometry is reported. Adducts of B[a]P-dihydrodiol epoxide (B[a]PDE) with guanosine and adenosine have been detected for the first time by use of precursor ion scan and neutral loss scan. B[a]P was then activated by use of UV irradiation and some of the products obtained have been identified by taking advantage of the information obtained for B[a]PDE. Photoactivation has also been carried out in the presence of hydrogen peroxide; this resulted in a higher yield of products with increased production of BaP diones. The reactivity of these compounds toward nucleosides has been tested. The proposed method was successfully used for detection of one stable guanosine-B[a]P dione adduct.

Multiclass analysis of illicit drugs in plasma and oral fluids by LC-MS/MS.

An analytical procedure for the simultaneous determination in human plasma and oral fluids of several illicit drugs belonging to different chemical and toxicological classes is presented. Amphetamine, methamphetamine, morphine, 6-monoacetylmorphine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylenedioxymethamphetamine, cocaine, benzoylecgonine, tetrahydrocannabinol, carboxytetrahydrocannabinol, ketamine, and phencyclidine have been quantified in real samples using a very rapid sample treatment, basically a protein precipitation. The quantitative analysis was performed by liquid chromatography-tandem mass spectrometry and has been fully validated. All the analytes were detected in positive ionization mode using a TurboIonSpray source, except carboxytetrahydrocannabinol, which was detected in negative ionization mode. The use of a diverter valve between the column and the mass spectrometer allows the preservation of the ion source performances for high-throughput analysis.

Evaluation of oxidized buckypaper as material for the solid phase extraction of cobalamins from milk: Its efficacy as individual and support sorbent of a hydrophilic-lipophilic balance copolymer.

This work describes a new analytical method for the determination of four cobalamins (adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), hydroxocobalamin (OHCbl) and cyanocobalamin (CNCbl)) in cow's milk. The extraction procedure is fast and based on dilution/protein precipitation of a milk sample with 50mM sodium acetate buffer (pH 4.6), followed by solid phase extraction (SPE) of the filtered supernatant. Relative recoveries higher than 60% have been obtained for all the cobalamins by combining two different types of sorbents in the same SPE cartridge: two disks of buckypaper (BP), a nanoporous felt composed of oxidized multiwalled carbon nanotubes (MWCNTs), separated by a Teflon frit from OASIS HLB (500mg), a hydrophilic-lipophilic balance copolymer. Before its use as sorbent, BP was characterized in terms of porosity, permeability, surface area, specific adsorption capacity and tested for a potential reuse after adequate chemical regeneration. The analysis of the extracts was performed by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) on an analytical C18 column in less than 10min. After validation, the method was applied to the determination of the natural content of the four B12 homologues in cow's milk samples, providing data lacking in the literature.

Solid-phase extraction followed by high-performance liquid chromatography-ionspray interface-mass spectrometry for monitoring of herbicides in environmental water.

In this work we developed a sensitive and specific multiresidue method, based on reversed-phase liquid chromatography-mass spectrometry, with an ionspray interface (LC-ISI-MS), for determining 52 of most representative compounds of herbicides in water samples. The procedure used involved passing 0.5 l of surface water, 2 l of ground water and 4 l of drinking water samples, respectively, through a 0.5 g graphitized carbon black (GCB) extraction cartridge. Base-neutral and acid herbicides were differential eluted from GCB cartridge and follow analyzed by HPLC-ISI-MS apparatus. A conventional 4.6-mm-ID reversed-phase LC C18 column, operating with a mobile phase flow-rate of 1 ml/min, was used to chromatograph the analytes. A flow of 100 microl/min of the column effluent was diverted to the ISI source. The study demonstrates the sensitivity of the technique, with detection limit under 10 ng/l in drinking water samples. Performance data for the method such as recovery and precision are also reported.

Solid-phase extraction followed by liquid chromatography-mass spectrometry for trace determination of beta-lactam antibiotics in bovine milk.

A confirmatory assay able to unambiguously identify and quantify 10 approved-for-use beta-lactam antibiotics in milk below stipulated U.S. and EU tolerance levels is presented. beta-Lactams are extracted from 10 mL of intact milk by a Carbograph 4 cartridge. After solvent removal, residue reconstitution, and filtration, a completely transparent and uncolored extract is injected into a liquid chromatography -mass spectrometry (LC-MS) instrument equipped with an electrospray (ES) ion source and a single quadrupole. During the chromatographic run, the ES/MS system is operated first in the positive-ion mode (PI) and then in the negative-ion (NI) mode. This is done to circumvent matrix interferences resulting in remarkable signal weakening of the last-eluted analytes, when detecting them as [M+H]+ adduct ions. MS data acquisition is performed by a time-scheduled three-ion selected ion monitoring program. At the 5 ng/mL level, recoveries of the beta-lactams are between 70 (nafcillin) and 108% (cephalin), with relative standard deviations ranging between 5 (oxacillin) and 11% (amoxicillin and ceftiofur). The response of the ES/MS detector is linearly related to injected amounts up to 500 ng, irrespective of the chemical characteristics of the beta-lactams and the acquisition mode selected (PI or NI modes). Limits of quantification, based on a minimal value of the signal-to-noise ratio of 10, were estimated to be within 0.4 (cephalin) and 3 ng/mL (dicloxacillin). Analyses of milk samples taken after intramammary application of amoxicillin showed that 1.2 ng/mL of this penicillin was still present 6 days after treatment. At this concentration level, the identification power of the method is not weakened, as signals of the three product ions of amoxicillin are still well distinguishable from the background noise.

Head-space chromatography in determination of enzymatic activity: microdetermination of the urinary kallikrein as arginine esterases.

Many enzymatic reactions yield volatile products either directly or by cascade sequences, so it seems possible that head-space chromatography might be used to determine enzymatic activity. The activity of urinary kallikrein, as arginine esterase, has been determined in this way by using the N(alpha)-acetyl-L-phenylalanyl-L-arginine ethyl ester as substrate and measuring the ethanol yielded on incubation for 10 min at 30 degrees, followed by quenching of the reaction. The method has been applied to aqueous solutions and urine.

Sample-pretreatment procedure for routine liquid chromatographic assay of serum cortisol.

A specific method for measuring concentrations of cortisol in serum, by preliminary isolation on a "minicolumn" followed by elution and determination by liquid chromatography, is described. This assay requires only 500 microl of serum. The limit of determination of cortisol was found to be 5 microg l . The analytical recovery of cortisol added to serum ranged from 98 to 102%. The coefficients of variation ranged from 2.4 to 3.4% (within-day) and from 3.6 to 8.8% (day-to-day), depending on the cortisol concentration. The method compares well with a commonly used radioimmunological method.

Solubility and thermal stability of some amino-mellitate compounds.

By reaction between the anion of mellitic acid (benzenehexacarboxylic acid) and some protonated linear polyamines (diethylenetriamine, triethylenetetramine, tetraethylene-pentamine, pentaethylenehexamine, spermidine, and spermine), fairly insoluble complexes have been obtained, with the general formula (amine)(x)(mellitate)H(6) (diethylenetriamine and spermidine, x=1; triethylenetetramine and spermine, x=0.75; tetraethylenepentamine, x=0.6 and 0.8; pentaethylenehexamine, x=0.5). K(s0) values for these complexes have been determined at I=0 mol dm(-3) and T=25 degrees C (logK(s0) ranges between -48.2 and -56.6). The solubility has been studied as a function of pH and of ionic strength. The thermal analysis, performed using air or argon flow, showed that all the solids behave in a similar way. In the range 20-120 degrees C the loss of hydration water occurs, and in the range 150-350 degrees C the first step of non oxidative decomposition takes place, with complete decomposition at 650 degrees C in air flow, whilst in argon flow the decomposition is still incomplete at 900 degrees C. Preliminary results of a parallel diffractometric study are also reported.

Total phosphorus determination in human bile. Comparison between two spectrometric methods.

The two most commonly used spectrometric methods for the determination of the phosphorus content of human bile are compared. The optimum experimental conditions are studied, and the analytical characteristics of the two methods, using both standard samples and human bile, are evaluated. The methods are compared on the basis of their sensitivity, precision and accuracy, and the correlation between the two techniques demonstrated using fifteen samples of human bile. Data obtained by both methods have been used to calculate lithogenic index values.

High-performance liquid chromatographic analysis of norfloxacin in human tissues and plasma with fluorescence detection.

A high-performance liquid chromatographic analysis with fluorimetric detection is described for the quantitative determination of norfloxacin in renal, prostatic tissues and in plasma. The analytical procedure in the tissue pretreatment, consists of purification of the obtained sample by a solid state extraction and quantitation by HPLC. The samples were chromatographed on a C(8) reversed-phase column. Analytical recoveries ranged from 95.2 to 97.6%. Within and between day precision were assessed by analysing serum containing 50 and 500 ng/ml norfloxacin. At each concentration, within day precision was < or = 3.6% (relative standard deviation, n = 10) and day-to-day precision was < or = 5.3% (n = 10). Limit of detection was ca 1 ng/ml.

Spongy state (status spongiosus) and inhibition of Na,K-ATPase: a pathogenetic theory.

Spongiform encephalopathies are characterized above all by spongiosis. This neuropathological characteristic is morphologically mimed by in vivo inhibition of cerebral brain Na,K-ATPase by means of subdural administration of ouabain. In this paper we underline the possibility that the 'spongiotic state' in other diseases might also be caused by the inhibition of this enzyme, thus hypothesizing that it is the enzyme itself that is targeted by the infective agent. The infective agent could, we believe, be shown to be linked to the enzyme if it were separated from the infected brain.

Comparison of extracting solutions for elemental fractionation in airborne particulate matter.

It is here described the comparison of extraction efficiency of some solutions (acetate buffer, deionized water, diluted HNO(3) and EDTA) frequently adopted in literature for evaluating the elemental solubility in airborne particulate matter. This comparison was performed considering the distribution of As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Mg, Mn, Na, Ni, Pb, S, Si, Sb, Sn, Sr, Ti, V, Zn between the extractable and mineralized residual fractions on the NIST 1648 certified material, PM(10) real samples and size-segregated samples, collected by a 13-stage impactor. The extracting solutions were evaluated by comparing extractive efficiencies and robustness towards some factors, such as acidity and concentration of complexing species, that have great environmental variability and that could be able to modify the extractive efficiency. Furthermore, extraction methods application to size-segregated samples allowed estimating the selectivity of extracting solutions towards dimensionally characterized emission sources, as dusts originated from abrasion and road dust re-suspension. On the basis of the obtained results, it was possible to define the main advantages and disadvantages resulting from the use of different extracting solutions, necessary to make possible the comparison of environmental studies carried out in different extractive conditions and to start up a proper study for harmonizing extracting procedures.

Applications of evolved gas analysis Part 1: EGA by infrared spectroscopy.

The analytical applications of the evolved gas analysis (EGA) performed by infrared spectroscopy, for the period extending from 2001 to 2004, are collected in this review. By this technique, the nature of volatile products released by a substance subjected to a controlled temperature program are on-line determined, with the possibility to prove a supposed reaction, either under isothermal or under heating conditions.

Applications of evolved gas analysis Part 2: EGA by mass spectrometry.

The analytical applications of the evolved gas analysis (EGA) performed by mass spectrometry, for the period extending from 2001 to 2004, are collected in this review. By this technique, the nature of volatile products released by a substance subjected to a controlled temperature program is on-line determined, with the possibility to prove a supposed reaction, either under isothermal or under heating conditions.

Thermogravimetry study on the water in secretory tissues of rabbit.

Isothermic and dynamic thermogravimetry was applied to rabbit parotid and submandibular glands to quantitate and characterize the water present inside them. Isothermic thermogravimetry demonstrated that at 95 degrees C a quota of water is still present in the samples. Dynamic thermogravimetry evidenced that temperatures higher than 200 degrees C are required for the release of the highest energy bound water from both glands. In addition during present research it was found that submandibular gland, an organ considered histologically homogenenous, reveals an heterogeneous distribution of water.

Differentiation of rat salivary glands by thermoanalytical analysis.

The water release from the sublingual, parotid and submandibular glands of male and female rats was analyzed by thermal analysis in order to detect the total water content and types. Different types of water, which are increasing from the sublingual to the parotid gland, were found and the relative distribution appeared to be a function of the bond energy of water to glandular components. In addition, evidence of a sexual dimorphism in the rat sublingual gland was demonstrated.

Characterization of mouse salivary glands by water content and type.

The thermoanalytical analysis was applied to samples of sublingual, submandibular and parotid glands from sexually mature mice of both sexes. Findings indicated that the three salivary glands show a behaviour of water release characteristic for each type of gland. Derivative thermogravimetry curves concerned with the sublingual and parotid glands belonging to male and female subjects exhibited overlapped results. As regards submandibular gland, instead, some differences emerged between subjects of different sex. Water content and types in sublingual, submandibular and parotid glands were discussed and related to the different morphological expression, histochemical reactivity and chemical composition of these organ tissues.

Thermal behaviour of the rabbit sublingual gland.

The purpose of this study was to show how some aspects of the biological matrix water can be investigated by thermoanalytical methods when using thermogravimetry and differential scanning calorimetry. Such methods allow to investigate the water in the rabbit sublingual gland and to quantitate it. The results supported the view that the water contained in this secretory organ show a large number of energetic interactions established on the basis of differential steps of water release by thermal disruption.

Determination of free fatty acids and lipase activity in milk: quality and storage markers.

The free fatty acid (FFA) content together with the lipase activity control can be considered as useful indexes of good quality and correct storage of food, especially for milk. The quantitative analysis of FFA in different kinds of milk has been performed by a potentiometric method, using a new extractive methodology outlined herein. The lipase activity has been controlled by a sensitive calorimetric method, previously validated by us, based on the direct measure of the heat quantity involved in the enzymatic reaction. In order to verify the milk quality after the healing treatments and/or during the shelf life, the behaviours of FFA content and of lipase activity have been outlined in function of storage time and pH variations on different typologies of milk. The FFA content in sample of fresh pasteurised milk was found to be quite high after the 5th/6th day of storage at +4 degrees C, meanwhile the pH values were always constant and only after the 9th day begun to decrease. At the same time the lipase activity, directly measured, was found to be appreciable after the 6th day of storage, giving an exothermic answer at the calorimeter, similar to that of a milk sample where only three international units of standard lipase were added.