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A liquid chromatography-tandem mass spectrometric with electrospray ionization (LC/ESI-MS/MS) method for determining the four naturally occurring aflatoxins (AFs) B1, B2, G1, and G2 in olive oil is proposed. AFs were extracted from oil sample by means of matrix solid phase dispersion (MSPDE), utilizing C18 as dispersing material. No further purification step, such as lipid removal, was performed. Aflatoxin M1, the hepatic metabolite of AFB1, was employed as internal standard. Olive oil extract was analyzed by LC/ESI-MS/MS in positive ionization mode, with multireaction monitoring acquisition. Due to a signal suppression ranging between 4 and 23%, quantitation was performed by matrix-matched calibration curves. The regression line coefficients of determination were above 0.9991. Sample recoveries ranged from 92 to 107%, with relative standard deviations below 13% for spiking levels between 0.5 and 5 ng g(-1); method quantification limits ranged between 0.04 and 0.12 ng g(-1). The developed LC/ESI-MS/MS method, although not as sensitive as LC coupled to fluorescence detection, is rapid, selective, accurate and precise, thus it can be used as confirmatory assay. The MSPDE appears suitable for application to other oleaginous matrices and for multiresidue investigation.

A solid phase extraction-high performance liquid chromatography–tandem mass spectrometry based analytical method suitable for simultaneous analysis of benzidine, 3,3′-dichlorobenzidine, mono-, di-, and tri-chloroanilines has been developed. Normal phase separation by liquid chromatography was performed using a cyano propyl methyl silica column, and atmospheric pressure photoionization was employed as interface with mass spectrometer. The developed method was evaluated in terms of limit of detection, accuracy, and precision. The quantification limit for all the compounds ranged between 7 and 112 ng L −1 , while recovery for all the compounds was higher than 94%. The method was tested by analyzing different industrial wastes, showing residual contamination by most of the analytes.

A rapid and sensitive procedure for determining residues of seven quinolone antibacterials in bovine muscle, kidney and liver is presented. The method is based on the matrix solid-phase dispersion (MSPD) technique with hot water as extractant followed by liquid chromatography/single quadrupole mass spectrometry (LC/MS) or triple-quadrupole mass spectrometry (LC/MS/MS). After dispersing tissue samples on hydrazine sulfate treated sand, target compounds were eluted from the MSPD column by passing through it 4 mL of water heated at 100 degrees C. After pH adjustment and filtration, 200 and 5 microL of the aqueous extracts were respectively injected into the LC/MS and LC/MS/MS instruments. With the former instrument, MS data were acquired in the three-ion selected ion monitoring mode, while MS/MS data acquisition was performed in the multi-reaction monitoring mode by selecting two precursor ion to product ion transitions for each target compound. Hot water appeared to be an efficient extracting medium, since absolute recoveries of the analytes were 84-102%. Using norfloxacin (a quinolone not used in veterinary medicine) as surrogate internal standard, the accuracy of the method at three concentration levels equal to 0.5, 1 and 1.5 times the maximum residue limits (MRLs) set by the european union was 88-109% with relative standard deviations (RSDs) not higher than 7%. The use of LC/MS/MS allowed detection and quantification of the analytes in any tissue considered to be performed at concentrations by far lower than half of their MRLs. Vice versa, the single-quadrupole MS arrangement, while succeeding in monitoring quinolones in muscle tissue at the 0.5 MRL level, showed to be not sufficiently selective for unambiguous identification of some quinolones in kidney and liver.

Anatoxin-a (AN) is a powerful neurotoxin that can be produced by cyanobacteria in eutrophic freshwaters. Consequently, AN can contaminate lakes, rivers and basins destined for drinking water and aquaculture. Two simple, specific and sensitive procedures for determining AN in lake water and fish muscle tissue are presented. Both analytical protocols are based on liquid-chromatography (LC)-tandem mass spectrometry (MS) with electrospray ionization. MS data were acquired in the multi reaction monitoring mode by selecting four precursor to product ion transitions. After filtration, AN in lake water was analyzed by directly injecting 0.5 ml of the aqueous sample in the LC column. Analysis of AN in fish muscle tissue involved the matrix solid-phase dispersion technique. The analyte was extracted from tissue by 4 ml of water acidified to pH 2 and heated at 80 degrees C. After acidification and filtration, 0.2 ml of the aqueous extract was injected in the LC column. Analyte recovery ranged between 71 and 79% and was not substantially affected by both the analyte concentration and the type of fish. Phenylalanine is an essential amino acid invariably present in any animal tissue. Like AN, this amino acid produces a pseudo molecular ion at m/z 166, it has a very similar fragmentation pattern and LC retention time. This method is able to prevent identifying phenylalanine for AN as the latter compound is eluted more than 1 min before the former one and the two compounds have remarkably different relative ion signal intensities. On the basis of a signal-to-noise ratio of 10, limits of quantification of AN in water and fish fillet were estimated to be 13 ng/l and 0.5 ng/g, respectively.

This report describes a liquid chromatography (LC)–tandem mass spectrometry (MS) multiresidue method for determining traces of 30 base/neutral/acid herbicides and fungicides in bovine whole milk. Four milliliters of milk was spiked with the analytes and two surrogate analytes (SAs) and then diluted with 35 mL of a water/methanol solution (50:50, v/v). This mixture was passed through a 0.5-g Carbograph 4™ cartridge. After washings, analytes were re-extracted by back-flushing the cartridge with 1.5 mL of methanol followed by 6 mL of methylene chloride/methanol (80:20, v/v), 50 mmol/L formic acid. After partial solvent removal down to about 0.1, 0.15 mL of 1 mmol/L formic acid aqueous solution and an internal standard (IS) were added. After filtration, 50 μL of the final extract was then introduced into the LC analytical column. During the chromatographic run, the MS system was operated in both positive and negative ion modes. MS data acquisition was performed in the multi-reaction monitoring mode, selecting two precursor ion > product ion transitions for each target compound, except for pentachlorophenol. On analyzing six milk samples from different sources, absolute recovery of the analytes and the two SAs ranged between 78% and 104% with RSDs not larger than 13%. The accuracy of the method at three different spike levels was assessed by adding the two SAs to analyte-containing milk samples and varied between 82% and 120% with RSDs not larger than 11%. Limits of quantification were estimated to range between 0.008 and 1.4 μg/L. Compared to the Carbograph 4 cartridge, one filled with a N -vinylpyrrolidone- m -divinylbenzene co-polymer (Oasis HLB™) sorbent was much less efficient in recovering several of the acidic herbicides considered and, in addition, its relative final extract produced a severe negative matrix effect that drastically weakened ion signal intensities of several non acidic analytes.

Microcystins (MCs) and cylindrospermopsin (CYL) are potent natural toxins produced by cyanobacteria (blue-green algae) that grow worldwide in eutrophic freshwaters and cause animal and human water-based toxicoses. The main purpose of this work has been assessing the contamination levels of some MCs and CYL in eutrophic Italian lake (Albano) water. To do this, we have developed an original analytical method involving MC extraction with a sorbent (Carbograph 4) cartridge. CYL is a highly polar compound that is scarcely retained by any sorbent material. To analyze this toxin, we directly injected 0.5 mL of filtered lake water into the liquid chromatography (LC) column. Analytes were quantified by LC coupled to tandem mass spectrometry in the multireaction monitoring mode. The recovery of five selected MCs added to an analyte free lake water sample at three different concentrations (50, 150, and 500 ng/L) ranged between 93 and 103% with RSD values no larger than 8%. Limits of quantification (LOQ) of the five MCs were within the 2-9 ng/L range, whereas the LOQ of CYL was 300 ng/L. The occurrence and abundance of cyanotoxins in Lake Albano was monitored over four months (Sept-Dec 2004) by analyzing water samples collected monthly at the center of the lake and at different depths (from 0 to -30 m). During survey and with the MS/MS system operating in the parent ion scan mode, we individuated two demethylated forms of MC-RR and one demethylated variety of MC-LR. Demethylated MC-RRs are known to be even more toxic than MC-RR toward zooplanktic grazers. CYL was the most-abundant toxin during the first three monitoring months. To the best of our knowledge, this is the first work reporting concentration levels of CYL in lake water.

Diastereomeric clusters of general formula [MAB 2 ] + and [MA 2 B] + (M = Li(I), Na(I), Ag(I), Ni(II)-H, or Cu(II)-H; A = (R)-(-)- and (S)-(+)-(1-aminopropyl)phosphonic acid; B = (1R)-(-)- and (1S)-(+)-(1-aminohexyl)phosphonic acid) have been readily generated in the electrospray ionization (ESI) source of a triple-quadrupole mass spectrometer and their collision-induced dissociation (CID) investigated. CID of diastereomeric complexes, e.g. [MA S (B S ) 2 ] + and [MA R (B S ) 2 ] + , leads to fragmentation patterns characterized by R h o m o = [MA S B S ] + /[M(B S ) 2 ] + and R h e t e r o = [MA R B S ] + /[M(B S ) 2 ] + abundance ratios, which depend upon the relative stability of the diastereomeric [MA S B S ] + and [MA R B S ] + complexes in the gas phase. The chiral resolution factor R c h i r a l = R h o m o /R h e t e r o is found to depend not only on the nature of the M ion but also on that of the fragmenting species, whether [MAB 2 ] + or [MA 2 B] + . The origin of this behavior is discussed.

A mass spectrometric method for extensive detection and semi-quantitative determination of flavonoid glycosides in stem and leaves of young Triticum durum plants is presented. About 100 g of sample were lyophilized and ground, and the compounds of interest were then extracted, cleaned-up, and fractionated using high-performance liquid chromatography (HPLC). Tandem mass spectrometry analyses were performed using a quadrupole-linear ion trap instrument with an information-dependent data acquisition (IDA) protocol that looped two experiments, enhanced MS scan and enhanced product ion scan. Various glycoconjugates, which are all derivatives of only four flavones, apigenin, luteolin, chrysoeriol and tricin, were identified and belong to the following categories: 7 monoglycosides, 31 diglycosides, 15 triglycosides and 1 tetraglycoside. Among these some acylated glycosides were found. Tricin derivatives are present exclusively as O-glycosides, while apigenin and luteolin are present always as C-glycosides. Semi-quantitative estimation was performed by using the monoglycoside and diglycoside of quercetin as internal standards.

An automated on-line solid-phase extraction-liquid chromatography-electrospray tandem mass spectrometry (SPE-LC-ESI-MS/MS) method was developed for the determination of ochratoxin A (OTA) in alcoholic beverages. Mean recoveries for wine and beer were, respectively, 75 and 82%. Detection was achieved in negative ionization with a Q TRAP mass spectrometer operating in multiple-reaction monitoring (MRM) mode or enhanced product ion (EPI) mode, using the third quadrupole as linear ion trap. The MRM mode turned out to be more sensitive; the method allowed accurate determination of OTA in the range of 0.01-25 ng mL(-1) using external calibration. Within-day and between-day relative standard deviation percentages were6.2 and9.1%, respectively. In EPI mode, fragmentation spectra at the limit of quantification (0.03 ng mL(-1)) and good linearity could be obtained. Application of the method (MRM mode) to the analysis of several wine and beer samples purchased in local stores revealed OTA levels in the ranges of 0.03-1.44 ng mL(-1) for wines and 0.02-0.14 ng mL(-1) for beers.

A sensitive, reliable liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for determining some commonly found mycotoxins produced by Fusarium strains in maize was evaluated and applied to field samples. The selected substances were: trichothecenes B (nivalenol, deoxynivalenol, fusarenon X, 3- and 15-acetyldeoxynivalenol) and some macrocyclic lactones (zearalenone, α- and β-zearalenol, zearalanone, α- and β-zearalanol). Analytes were extracted from a 1 g sample by homogenization with acetonitrile/water (75:25, v/v, 25 mL final volume). 5 mL of crude extracts was cleaned-up on Carbograph-4 cartridges. Two fractions were obtained and were analyzed by HPLC-electrospray ionization (ESI) in negative mode. Recoveries for spiked maize samples were in the range 79–106% and method detection limits (MDLs) were ⩽6 ng/g for all compounds, except fusarenon X (12 ng/g). 25 random maize samples were analyzed both by the ELISA-based methods specific for deoxynivalenol and zearalenone and by this method for trichothecenes B and macrocyclic lactones. Results were comparable for zearalenone ( R 2 = 0.982), but disagreed for deoxynivalenol. Finally, a total of 78 freshly harvested maize samples, collected from central and northern Italy during 2002, and divided in two different experiments, were analyzed by the developed method. Data show that there exists a phenomenon of random contamination from the target fusariotoxins just before harvest and an increase of trichothecene B and zearalenone abundance on field crop possibly related to damp climate, temperature range and delayed harvest period. Deoxynivalenol was the most abundant (up to 3430 ng/g) and frequent mycotoxin (40%) detected, followed by acetyldeoxynivalenol. Derivatives of zearalenone were present in traces and β-zearalanol was never found.

Phytoestrogens activate a biological response in vertebrata where they can mime or modulate the action of endogenous estrogens. For this reason they have been subjected to several studies about their physiological effects on humans and many analytical methodologies for their determination in food matrices and physiological fluids have been developed. On the contrary, little information can be found in literature about the presence of isoflavones and coumestrol in the environment, even if it is known that this may have significance. In the present study we investigated the presence of nine selected free and conjugated phytoestrogens in environmental water. A liquid chromatography–mass spectrometry (LC–MS/MS) based analytical methodology was developed and employed for detection of target compounds in surface water and wastewater. The methodology uses solid-phase extraction, followed by high performance liquid chromatography coupled to tandem mass spectrometry using an electrospray (ESI) interface operating in positive ion mode (LC–ESI–MS/MS). The extraction was made with 200 mg, 6 mL OASIS HLB® cartridges. Recoveries for the selected compounds were in the 67–97% range for all the considered analytes. The method was employed for environmental monitoring. Samples of river water and wastewater collected over a 4-month period were analyzed with the developed procedure. Results showed the presence of isoflavones in most of the samples analyzed. Average concentration of target analytes found in wastewater sewage treatment plant influent ranged from 454 to 12 ng/L. In effluent water and river water the analytes were present at lower concentration.

A simple, selective and sensitive procedure for determining nine widely used aminoglycoside antibiotics (AGs) in bovine whole milk is presented. It is based on matrix solid-phase dispersion with heated water, at 70 degrees C, as extractant followed by liquid chromatography (LC)-tandem mass spectrometry (MS) using an electrospray ion source. After acidification and filtration, 0.2 ml of the aqueous extract was injected into the LC column. MS data acquisition was performed in the multi reaction monitoring mode, selecting two (three, when possible) precursor ion > product ion transitions for each target compound. Analyte recoveries ranged between 70 and 92%. Using aminosidine (an AG not used in veterinary medicine) as surrogate internal standard, the accuracy of the method at three spike levels varied between 80 and 107% with R.S.D. not larger than 11%. The limits of quantification were between 2 ng/ml (apramycin) and 13 ng/ml (streptomycin). They are well below the tolerance levels set by both the European Union and the U.S. Food and Drug Administration.

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determining the type B fumonisin mycotoxins in corn-based foodstuffs is described. Fumonisins FB1 and FB2 were extracted from a 1 g sample by homogenization with acetonitrile/water (75:25, v/v, 50 mmol/L formic acid, 25 mL final volume) and the extract was defatted on C18 phase. Volumes of 5 mL of crude extracts were cleaned up on Carbograph-4 cartridges. The final solution was analyzed by HPLC with electrospray ionization mass spectrometry in positive ion mode using multiple reaction monitoring with a QqQ linear ion trap mass spectrometer. Recoveries for spiked corn-based foodstuffs ranged from 91-105% (RSD%or =8%), and method detection limits wereor =2 ng/g for FB1 andor =1 ng/g for FB2. Two different spiking levels were tested (5000 and 100 ng/g for FB1, 1000 and 20 ng/g for FB2). Quantitation was achieved by an external calibration procedure using matrix-matched standards, with diclofenac added post-cleanup as internal standard for the LC/MS/MS analyses. Calibration curves showed linearity in the concentration range 0.005-5 ng/microL of final extract (0.992or = R2or =0.995). Two other fumonisins, FB3 and FB4, were identified in naturally contaminated samples of corn meal using an information-dependent acquisition protocol that looped three experiments, including neutral loss scan, enhanced resolution scan, and enhanced product ion scan. FB3 and FB4 quantitation was estimated as peak area ratios relative to the FB2 response in view of the lack of both standards. This work also includes an application of the present LC/MS/MS method to some maize and maize-based product samples (corn meal, cornflakes and popcorn) collected from Italian stores. FB1 and FB2 contamination levels exceeding the European Union recommendation were found in 8 out of 15 corn meal samples.

A simple, specific and rapid procedure for determining six largely used carbamate insecticides in bovine whole milk is here presented. This method is based on the matrix solid-phase dispersion technique with heated water as extractant followed by liquid chromatography (LC)-mass spectrometry (MS) equipped with a single quadrupole and an electrospray ion source. Target compounds were extracted from milk by water heated at 90 degrees C. After acidification and filtration, 0.2 mL of the aqueous extract was injected in the LC column. MS data acquisition was performed in the selected ion-monitoring mode, selecting three ions for each target compound. Heated water appeared to be an excellent extractant, since absolute recovery data ranged between 76 and 104% with R.S.D. not larger than 8%. Using butocarboxim (an obsolete carbamate insecticide) as surrogate internal standard, the accuracy of the analysis at three spike levels varied between 85 and 105% with R.S.D. not larger than 9%. On the basis of a signal-to-noise ratio of 10, limits of quantification were estimated to range between 3 ppb (propoxur) and 8 ppb (pirimicarb). The effects of temperature, volume and flow rate of the extractant on the analyte recovery were studied.

Phytoestrogens are diphenolic compounds that are present in several edible plants and are particularly abundant in soybeans. Because of their estrogenical, antriestrogenical, anticarcinogenic and antioxidant activities in animal and humans, they became of great interest. Dietary factors are considered important in determination of risks, in fact, studies have revealed beneficial or protective effects of the consumption of vegetables, in particular soy and soybean products. So that in the present paper the simultaneous determination of eight isoflavones and coumestrol in vegetables is reported. The quantitative analysis has been made by means of LC separation combined with tandem mass spectrometry. In particular, a new simple and fast extraction methodology and a clean-up, based on cold aided de-fatting, is proposed. Method performance was evaluated by comparison with a reference procedure. The developed procedure was then used for a survey of phytoestrogens concentration in some selected vegetables.

The kinetic method has been used for enantiodiscriminating important chiral-residues from post-translationally modified proteins, i.e., the O-phospho alpha-amino acids, and some chiral a-amino acid mimics, i.e., the alpha-aminophosphonic acids. Their proton affinities (PA) have been evaluated using the same method in its extended form which allows the estimate of the entropy contributions on the kinetic measurements. The kinetic approach is based on the competing collision-induced decomposition (CID) of suitable proton-bound complexes between the P-containing molecules and suitable reference compounds, e.g., a-amino acids. The investigation was carried out in a QqQ instrument as a function of the collision energy (4-14 eV). The proton-bound complexes were generated in an electrospray ionization (ESI) source from calibrated water/methanol solutions of the two components. The experimental results have been discussed and compared with theoretical calculations. (C) 2003 Elsevier Science B.V. All rights reserved.

Identification and quantification of the enantiomeric excess of O-phosphoserine (

Clusters between first-group metal ions and chiralα-aminophosphonic acids have been readily generated by Pulsed Laser Ablation (PLA) and by Electrospray Ionization (ESI) and their fragmentation investigated by mass spectrometry. The complexes studied have the general formula[Me(I)Cl2]+, where Me(I) is H, Li, Na, or K, C is (R)-(—)-(1-aminoethyl) phosphonic acid(ER)and (S)-(+)-(1-aminoethyl) phosphonic acid(ES),(1R)-(+)-(1-amino-2-methylpropyl) phosphonic acid(PR)and (1S)-(—)-(1-amino-2-methylpropyl) phosphonic acid(PS),(1R)-(-)-(1-amino-hexyl) phosphonic acid (HR) and (1S)-(+)-(1-amino-hexyl) phosphonic acid (HS), o-phospho-L-serine (SS)ando-phospho-D-serine(SR), and L is a referenceα-aminophosphonic acid (E, P, H or S) of defined configuration. Collision induced dissociation (CID) of diastereomeric[Me(I)Cl2]+complexes leads to fragmentation patterns characterized by[Me(I)Cl]+/[Me(I)L2]+abundance ratios which depend upon the configuration of solute C. These different spectral features were correlated to the different stability of the diastereomeric[Me(I)CRL]+and[Me(I)CSL]+complexes in the gas phase.

A multiresidue analytical method for the determination of the most common and biologically active natural and synthetic steroids (four estrogens: estriol, 17β-estradiol, 17α-ethynylestradiol, estrone; one progestagen: progesterone and six androgens: trenbolone, boldenone, nandrolone, testosterone, 17α-methyltestosterone, stanozolol) in environmental waters was developed. The analytes were isolated from water samples by solid phase extraction (SPE) utilizing a graphitized carbon black adsorbent (Carbograph-1). The final samples were analyzed by reversed-phase high performance liquid chromatography with tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS-MS). Ionization was performed in a heated nebulizer (HN) interface operating in the positive ion mode. The protonated ions [M+H]+ and the dehydrated ions [M+H-H2O]+ (for estriol, 17α-estradiol, 17β-estradiol, and 17α-ethynylestradiol) were used as precursor ion for collision-induced dissociation (CID), and two diagnostic prod...

A method for the identification and quantification of neutral diphenyl-ether (DPhE) (aclonifen, bifenox, fluoroglycofen, lactofen, oxyfluorfen) and acid metabolites (acifluorfen, bifenox acid, fomesafen) at a low nanogram per liter concentration level in natural water is presented. These herbicides and their metabolites were determined in drinking water, groundwater, and river water. The analytes were isolated from water samples by solid-phase extraction (SPE) on Carbograph-1 cartridges and analyzed with reversed phase high-performance liquid chromatography (RP-HPLC) using UV detection at a wavelength of 290 nm. The isolation procedure separated the acid DPhE from neutral DPhE during the elution from Carbograph-1 cartridge using dichloromethane:methanol (80:20, v/v) for neutral compounds and dichloromethane:methanol (80:20, v/v) acidified with 25 mmol/l of formic acid for the anionic metabolites. Quantification was performed by generation of an external calibration curve. High recoveries (>85%) of extraction were obtained for all the compounds. The method detection limit ranged from 4.1 to 8.7 ng/l for drinking water, from 5.1 to 9.5 ng/l for groundwater and from 17.5 to 36.2 ng/l for river water. This method involves confirmatory analysis by tandem mass spectrometry (MS-MS) in selected reaction monitoring (SRM) mode. Conditions for MS-MS detection of characteristic daughter ions formed by collision-induced dissociation of the parent ion are described. The final samples were analyzed by HPLC–MS-MS utilizing a heat-assisted electrospray interface (turbo ion spray) (TISP) for acid DPhE and a heated nebulizer (HN) interface for neutral DPhE. Quantification was performed by generation of an internal calibration curve. Excellent method precision was demonstrated with a relative standard deviation of less than 18% for all analytes at all concentration levels. Application of the method for detecting DPhE herbicide residues in a real-world groundwater samples was demonstrated.

A multiclass method for the determination of five selected sulfonylurea, diphenylether, and sulfonanilide herbicides commonly used in maize growing has been developed. In this study, the method development for the analysis was performed using a solids extraction unit for analyte extraction from maize. A procedure using solid phase extraction system for clean up the methanolic extract, and a liquid chromatography-tandem mass spectrometry (MS/MS) was developed for the final determination. The extraction parameters that were studied included temperature, duration of the static extraction, sample amount, solvent volume, and flow rate. Maximum yield was obtained by extraction with 25 mL of pure methanol at 50 °C and a flow rate of 1 mL min−1. After extraction, herbicides present in methanolic phase were isolated and cleaned up by solid phase extraction using a reversed-phase C-18 cartridge and Carbograph-1 cartridge. The herbicides were chromatographically separated using reversed-phase HPLC and selectively analyzed by MS/MS using a positive ionization-turbo ion spray (PI-TISP) interface in multiple reaction monitoring (MRM) mode.

This paper describes the simultaneous quantification of the imidazolinone herbicides (IMIs) imazapyr, m-imazamethabenz, p-imazamethabenz, m,p-imazamethabenz-methyl, imazethapyr, and imazaquin in two types of samples. (a) Groundwater, lake water, and river water samples were enriched by off-line solid-phase extraction with a Carbograph-1 cartridge and analyzed by reversed-phase liquid chromatography using a UV detector (lambda = 240 nm). The overall recoveries of IMIs extracted from 1 L of groundwater (fortified with 500-100 ng/L), 0.5 L of lake water (fortified with 500-100 ng/L), and 0.5 L of river water (fortified with 1000-200 ng/L) samples were not lower than 89%. The mean relative standard deviation (RSD) was 5.1% (ranging from 4.1% to 6.8%) in natural water. The detection limits were 30-39 ng/L in groundwater, 43-51 ng/L in lake water, and 55-67 ng/L in river water. The method involves confirmatory analysis by LC/ES-MS in full-scan mode. The dependence of the ion signal intensities on proton concentration in the mobile phase was investigated with a view to optimizing the sensitivity of the ES-MS detector. When LC/ES-MS was used, the limit of detection, calculated from extracted-ion current profiles (EICPs), was 4-7 ng/L for ground-water and 9-13 ng/L for river water. (b) Soil sample analysis utilized combined soil column extraction (SCE) and off-line solid phase extraction (SPE) for sample preparation, analyzing with LC/ES-MS under selected ion monitoring (SIM). Several different extractants were evaluated for the purpose of SCE optimization. The system that best optimizes the extractability IMIs from the soil was found to be the mixture CH3OH/(NH4)2CO3 (0.1 M, 50:50 v/v). The effect of IMI concentration in the matrix on recovery was evaluated. The total recovery of each IMI from soil at each of the two levels investigated ranged from 87% to 95%. Under three ion SIM conditions, the limit of detection (S/N = 3) was 0.1-0.05 ng/g in soil samples.

When nanoparticles (NPs) enter a physiological environment, medium components compete for binding to the NP surface leading to formation of a rich protein shell known as the "protein corona". Unfortunately, opsonins are also adsorbed. These proteins are immediately recognized by the phagocyte system with rapid clearance of the NPs from the bloodstream. Polyethyleneglycol (PEG) coating of NPs (PEGylation) is the most efficient anti-opsonization strategy. Linear chains of PEG, grafted onto the NP surface, are able to create steric hindrance, resulting in a significant inhibition of protein adsorption and less recognition by macrophages. However, excessive PEGylation can lead to a strong inhibition of cellular uptake and less efficient binding with protein targets, reducing the potential of the delivery system. To reach a compromise in this regard we employed a multi-component (MC) lipid system with uncommon properties of cell uptake and endosomal escape and increasing length of PEG chains. Nano liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) analysis allowed us to accurately determine the corona composition showing that apolipoproteins are the most abundant class in the corona and that increasing the PEG length reduced the protein adsorption and the liposomal surface affinity for apolipoproteins. Due to the abundance of apolipoproteins, we exploited the "protein corona effect" to deliver cationic liposome-human plasma complexes to human prostate cancer PC3 cells that express a high level of scavenger receptor class B type 1 in order to evaluate the cellular uptake efficiency of the systems used. Combining laser scanning confocal microscopy with flow cytometry analysis in PC3 cells we demonstrated that MC-PEG2k is the best compromise between an anti-opsonization strategy and active targeting and could be a promising candidate to treat prostate cancer in vivo.

The reliability of total sialic acid (TSA), lipid sialic acid (LSA) and free sialic acid (FSA) as markers in genitourinary malignancies was evaluated in 20 normal subjects, 21 patients with prostatic cancer, 22 patients with urinary bladder cancer and 14 patients with renal cell carcinoma. We introduce the new concept of 'corrected' lipid sialic acid (CLSA), which expresses the actual concentration of sialic acid bound to glycolipds by subtracting the concentration of FSA determined by a novel ultrafiltration method. TSA did not show significant differences with respect to normal controls, except for renal cell carcinoma, whose mean value (879 +/- 145 micrograms/ml) showed a P value < 0.001. Instead, CLSA showed only significant differences (P = 0.001), with respect to normal controls in stage I and in all grades of renal cell carcinoma. While all data indicated significant increases (P = 0.001) in the FSA values, (means +/- S.D.) of 0.621 +/- 0.272 micrograms/ml were found in patients with prostatic cancer, 0.796 +/- 0.443 micrograms/ml in patients with urinary bladder cancer and 0.667 +/- 0.146 micrograms/ml in patients with renal cell carcinoma. Separate TSA and CLSA measurements appeared to be of limited value in the detection of genitourinary malignancies. However, results show that FSA was the most sensitive of the three markers tested for detecting malignancies.

A rapid, sensitive and specific assay was developed for routine determination of melatonin in human serum. Melatonin was measured by liquid chromatography with fluorescence detection (λex = 285 nm, λem = 345 nm), using an isocratic mobile phase consisting of water-acetonitrile (75:25, v v ) . A simple “solid phase extraction” procedure employing LC-18 and Carbograph as adsorbents was used to isolate and purify the compound of interest from serum samples. The average recovery was 88.9% (range 86.3–91.7%). The method gave satisfactory reproducibility and accuracy, the detection limit of the system was 2 pg for injection and the practical limit of the assay for serum is ca. 3 pg ml−1 for 2 ml of sample. In order to evaluate the specificity, the potential interference of 33 compounds (chiefly monoamines) arising from the metabolism of tyrosine and tryptophan were studied. The melatonin concentration in 21 serum samples measured in the present study was compared by using an radioimmunoassay method. Some results on the melatonin circadian rhythm are presented to illustrate the applicability of this method.

A method has been developed for the determination of thirteen phenylurea herbicide residues in milk. It involves one-step solvent extraction of the milk with methanol by ultrasonication. The extract is cleaned up on an Amberchrom resin cartridge. Reversed-phase, gradient elution, high-performance liquid chromatography with UV detection at 242 nm is used to analyse the residues. The recovery of thirteen phenylurea pesticides is quantitative, ranging from 71.4% to 97.9% for the individual herbicides investigated at concentrations around 0.05 mg kg−1 and from 65.1% to 95.6% around 0.005 mg kg−1. The method is not associated with any of the emulsion problems common to conventional solvent extraction, which considerably reduce the sample clean-up process compared with existing methods.

A rapid, sensitive and selective high-performance liquid chromatographic method has been developed for the determination of sphingosine in human serum. After precipitation with methanol, the samples were extracted using Carbopack B disposable columns; the sphingosine was eluted with 0.05 M hydrochloric acid in methanol-dichloromethane (20∶80, v/v) and the extract evaporated to dryness at 40°C. The sample residue was then reconstituted with methanol and reacted with o-phthaldialdehyde reagent to produce a fluorescent compound. Separation was performed using an LC-18 column with 0.05 M phosphate buffer (pH 7)-methanol-acetonitrile (15∶80∶5, v/v) as mobile phase. Fluorescence detection was performed with excitation and emission wavelengths of 340 and 455 nm, respectively. The serum extract was re-analyzed with a cyano LC column to minimize the possibility of false positive results. The possible interference of compounds having a structure similar to that of sphingosine was evaluated. The mean recovery of sphingosine was >94.5%. The limit of detection of the assay was 1 ng mL−1. The between-run and within-run coefficients of variation for replicate analyses were

Here we investigate the effect of membrane charge density on the protein corona that forms upon exposure of cationic liposomes to human plasma. We show that the protein corona around cationic liposomes is not uniquely defined by membrane charge density, but it is deeply affected by both the overall cationic charge and the available surface area. Remarkably, changes in protein composition are largely removed when a correcting quantity accounting for the ratio between the cationic charge and the surface area of different cationic liposome formulations is considered.

The mechanism of cell uptake in lipid mediated gene delivery was investigated in NIH3T3 and CHO cell lines. We show that different endocytic pathways are activated by shape coupling between lipoplex and membrane lipids. Our results suggest that tailoring the lipoplex lipid composition to the patchwork-like plasma membrane profile could be a successful machinery of coordinating the endocytic pathway activities and the subsequent intracellular processing. Transfection experiments performed at 4°C, when endocytosis does not take place, show that a novel class of highly efficient multicomponent lipoplexes enter cells by a temperature-independent fusion-like mechanism. In vivo, plasma proteins bind to lipoplex surface and create a rich ‘protein corona’ that is recognized by cells and other biological structures. The ‘protein corona’ associated to lipoplexes after interaction with human plasma was found to be much richer in basic immunoglobulins gamma proteins (Ig-Gs) than that of pure lipid vesicles in the absence of DNA. Because surface properties of lipoplexes may determine their interaction with cells and tissues, an accurate knowledge of lipoplex surface properties may be important for predicting biological responses. These findings also suggest the existence of hybrid structures made of multilamellar complexes either stuck together by DNA or coexisting with DNA-loaded intact vesicles.

A simple, rapid, and extremely sensitive method for determining N -acetyl- and N -glycolylneuraminic acids in serum and in submandibular, sublingual, and parotid glands using high-performance liquid chromatography with a fluorometric detector is described. The neuraminic acids contained in the samples are released in the presence of 2 M CH 3 COOH by means of microwave hydrolysis (only 10 min required) and are subsequently converted using 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for α-ketoacids, into highly fluorescent derivatives. In order to optimize the release of sialic acids and to minimize the effect of destruction, the following analytical variables were investigated: temperature, time, and concentration of the acid. Within 12 min after derivatization, the compounds were separated on a reversed-phase column by means of isocratic elution using a mobile phase of water (pH 3 with H 3 PO 4 )-methanol-acetonitrile (86:6:8, v/v). Fluorescence detection was performed at an excitation wavelength of 373 nm and an emission wavelength of 448 nm.

The amounts of free sphingosine in Epstein Barr virus tranformed B lymphocytes (EBV-B) treated with sphingosine and phorbol-12,13-dibutyrate (PD) has been quantified by high performance liquid chromatography (HPLC). PD treatment did not affect intracellular sphingosine level, while it seems to lessen the removal of this long chain base in sphingosine-treated EBV-B cells. The previous results relative to sphingosine-dependent changes in choline-metabolite levels have to be interpreted on the basis of these results.

The problem of the maturing of thermal mud has long been a subject of discussion by many workers, without any objective solution being found. The process consists of the interaction between a fluid component (mineral water) and a solid component (virgin mud). Maturing is an extremely complex phenomenon which includes not only processes peculiar to the interaction between these components but also a wide range of biochemical processes related to the growth of algae and the micro-organisms characteristic of thermal mud. These processes are influenced by the ecosystem in which maturing takes place. Maturing involves a complex set of modifications which can be summed up as: (1) modifications related to the virgin mud, (2) appearance of new chemical species or an increase in those already present, and (3) disappearance or decrease in chemical substances already present. In the present paper, maturing has been investigated using thermal analysis, reflectance spectroscopy. X-ray diffraction, gas, liquid and ionic chromatography, atomic absorption and nuclear magnetic resonance. Families of molecules and physico-chemical parameters have been analysed to evaluate modifications as a function of time, with the aim of identifying any trends in such modifications which might lead to equilibrium conditions which could be considered indicative of maturing.

Identification and quantification of the enantiomeric excess of O-phosphoserine (OP Ser) residues from post-translational modified proteins is now possible by the application of a very reproducible and sensitive mass spectrometric method. This technique is based on the different mass-spectral fragmentation patterns of the diastereomeric cluster ions generated in water/methanol solutions of the analyte (OP SerD/L ) with a suitable chiral compound (OP ThrL ) by collision-induced dissociation (CID; see mass spectra).

A method for the extraction and quantification of benzydamine and its metabolite N-oxide by liquid chromatography with fluorescence detection in plasma samples is described. This method has adequate sensitivity, specificity and is reproducible. The use of the extraction column allowed a recovery of both benzydamine and its metabolite of over 97% to be obtained. The plasma levels of benzydamine and its metabolite N-oxide were studied after oral administration as sugar-coated tablets or topical application to the vaginal mucosa as a cream to 6 healthy volunteers. After topical application, the plasma concentrations of the unchanged drug and its metabolite are lower than those obtained following oral administration. These data further stress the concept that, whenever possible, topical use should be considered the treatment of choice since, along with a more selective therapy, the incidence of systemic side effects can be considerably reduced.

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 Nα-acetyl- l -phenylalanyl- l -arginine ethyl ester as substrate and measuring the ethanol yielded on incubation for 10 min at 30°, followed by quenching of the reaction. The method has been applied to aqueous solutions and urine.

The application of graphitized carbon black to the extraction of traces of organic acids has been studied. It is demostrated that the recovery of these substances cannot be carried out without considering the nature of these compounds and their molecular structure. The amount of adsorbent and the kind of eluent required were deduced respectively from the breakthrough values and from distribution ratios.

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 ⩽3.6% (relative standard deviation, n = 10) and day-to-day precision was ⩽5.3% ( n = 10). Limit of detection was ca 1 ng/ml.

Chromatography on Bio-Gel P-2 and high-performance liquid chromatography on RP-18 columns monitored with UV, fluorescence and electrochemical detectors have been used to evaluate the efficiency of granular, uncoated, active charcoal to remove from the ultrafiltrates of uraemic patients those organic substances accumulated in the blood that are not easily removed by dialysis. Chromatography on Bio-Gel P-2 and high-performance liquid chromatography on RP-18 columns carried out isocratically and monitored with an electrochemical detector seem very useful for clinical investigation as they increase the information obtained from routine haematochemical analyses such as blood urea nitrogen, creatinine, uric acid and electrolytes (calcium, phosphorus, sodium and potassium).

Analysis for 13 common organophosphorus pesticides by thin-layer chromatography is described; 17 solvent systems were examined. With channel thin-layer chromatography, linear calibration graphs were obtained for the range 1–10 μg.

Site localization of protein sulfation by high-throughput proteomics remains challenging despite the technological improvements. In this study, sequence analysis and site localization of sulfation in tryptic peptides were determined under a conventional nano-liquid chromatography-mass spectrometry configuration. Tryptic sulfopeptide standards were used to study different fragmentation strategies, including collision-induced dissociation (CID), higher-energy collisional dissociation (HCD), electron-transfer dissociation (ETD), electron-transfer/higher-energy collision dissociation (EThcD), and electron-transfer/collision-induced dissociation (ETciD), in the positive ionization mode. Sulfopeptides displayed only neutral loss of SO

A method is proposed for the determination of six polycyclic hydrocarbons in water samples, using a short column packed with graphitized carbon black (GCB). The adsorbed compounds are eluted by passing through the column toluene-benzene-acetonitrile (5:2:3). In order to choose the optimal eluent, the partition coefficients in GCB-liquid systems were studied in a static arrangement; to optimize the size of the concentration columns breakthrough curves were plotted.

The problem of water content modification in tissues, induced by Dupuytren's disease, is treated by differential scanning calorimetry (DSC) and thermal analysis. Two different types of water are found in the pathological tissue instead of the only one found in healthy tissue. At the same time quantitative analysis shows an increase in the total water in the pathological tissue. Finally, the ratio of the heights of the two maxima found in the pathological tissue is proposed as a diagnostic parameter for the evaluation of the development of the pathology.