Carlo Scapolla

Rapid and selective determination of UV filters in seawater by liquid chromatography-tandem mass spectrometry combined with stir bar sorptive extraction.

Fast liquid chromatography coupled to triple-quadrupole tandem mass spectrometry was employed for the determination of six UV filters in seawater. The separation of the analytes was achieved in less than 5 min; polarity switching was used as four of the analytes were ionized in positive mode and the remaining two in negative mode. Two ionization sources were employed and compared: atmospheric pressure chemical ionization (APCI) gave better results than electrospray ionization (ESI) for all analytes, with higher reproducibility and lower detection limits. Therefore APCI was chosen for the determination of the analytes in seawater samples using stir bar sorptive extraction-liquid desorption (SBSE-LD). Quantitative analysis was performed in multiple reaction monitoring (MRM) mode; fragmentation pathways of the analytes with regard to the formation of the MRM ions were also proposed. For the analysis of seawater samples, calibration curves were drawn using SBSE in spiked seawater. All figures of merit of the method were satisfactory; limits of detection were particularly low for the four analytes ionized in positive mode, being in the range 8-31 ng/L. The method was applied to the determination of the six UV filters in seawater samples from Liguria, Italy. Only benzophenone-3 (BP-3) and ethylhexyl methoxycinnamate (EHMC) were measured in the analyzed samples; some of the remaining analytes were also detected but always below the limit of quantitation.

An improved method for simultaneous analysis of aminothiols in human plasma by high-performance liquid chromatography with fluorescence detection

Altered levels of aminothiols in biological fluids are thought to be an important risk indicator for several diseases, and reliable methods for the accurate determination of aminothiols concentrations in plasma are thus required. In this paper ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-BF) is proposed as a convenient fluorogenic derivatizating reagent for the determination of aminothiols (cysteine, cysteinylglycine, homocysteine and glutathione) by HPLC with fluorescence detection. The reactions of SBD-BF with aminothiols at room temperature are about three-times faster than those of ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (the most frequently employed reagent) at 60 degrees C. The derivatives of SBD-BF with cysteine, cysteinylglycine, homocysteine and glutathione are easily separated by HPLC and their calibration curves show excellent linearity over the range 0.05-20 micromol/L with excellent r(2) values for all analytes. SBD-BF reacts with thiols under mild conditions, i.e. at 25 degrees C over about 30 min, and is proposed as a suitable fluorogenic reagent for thiol derivatization to be introduced in analytical clinical chemistry. The detection limits of Cys, Cys-Gly, Hcy and GSH at a signal-to-noise ratio of 5 were 0.1 microM for Cys, 0.01 microM for Cys-Gly and Hcy, and 0.02 microM for GSH. Furthermore, validation parameters of the proposed method are quite satisfactory. As an application of this method the determination of thiol derivatives in human plasma was carried out on a number of samples.

Emerging pollutants in aquatic environments: monitoring of UV filters in urban wastewater treatment plants

Six UV filters – benzophenone-3 (BP-3), octocrylene (OC), ethylhexyl dimethyl p-aminobenzoate (OD-PABA), ethylhexyl methoxycinnamate (EHMC), ethylhexyl salicylate (EHS) and homosalate (HMS) – with endocrine disrupting potential were monitored in different wastewater treatment plants (WWTPs) located in Genoa, Italy. The influent and effluent samples were collected once a month from April to September 2011. The analytes were determined by stir bar sorptive extraction followed by liquid desorption (SBSE-LD) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantitative analysis was performed in triggered MRM (tMRM), which allowed improvement of specificity without compromising sensitivity. In the inlet samples four analytes were detected; in particular BP-3, OC, EHMC, and OD-PABA were in the range of 4–163, 12–390, 23–68, and 2–4 ng L−1, respectively. Measured concentrations indicated variability of UV filter inputs to WWTPs, with higher loads during the warmer months. A highly positive correlation was found between air temperature and the measured concentration of OC and BP-3. Only BP-3 and OC were detected in some effluent samples, with considerably lower concentrations. The removal efficiencies of the plants were in the range of 64 to >99% and 94 to >99% for BP-3 and OC, respectively.

Determination of endocrine-disrupting compounds in drinking waters by fast liquid chromatography-tandem mass spectrometry.

Growing attention has been recently paid to safety of food and drinking water, making necessary the adoption of policies for water sources protection and the development of sensitive and rapid analytical methods to identify micropollutants. Endocrine-disrupting compounds (EDCs) have emerged as a major issue as they alter the functioning of the endocrine system. Since ingestion of EDCs via food is considered the major exposure route, there is a growing interest in understanding EDC fate during drinking water treatment and in monitoring potential contamination of surface waters and groundwaters. In this work, a fast liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for the determination of 4-n-nonylphenol (NP), bisphenol A (BPA), estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) in drinking waters. In the literature analytical articles seldom provide details regarding fragmentation pathways. In this paper spectra of the five EDCs in negative ESI were interpreted with the support of accurate mass spectra acquired by a quadrupole time-of-flight instrument; fragmentation pathways were also proposed. The chromatographic separation of EDCs was optimized on a Pinnacle DB Biphenylic column with a water-acetonitrile gradient. Quantitative analysis was performed in multiple reaction monitoring (MRM) mode using bisphenol A-d(16) (BPA-d(16)) as internal standard; calibration curves showed good correlation coefficients (0.9989-0.9997). All figures of merit of the method were satisfactory; limits of detection were in the range 0.2-0.4 ng/ml. The method was applied to the determination of the analytes in waters sampled by polar organic chemical integrative samplers in a drinking water treatment plant. Rather low concentration of BPA, NP and E1 were measured in the inlet, while none of the considered EDCs was detected in the outlet.

A simple, sensitive and efficient assay for the determination of d- and l-lactic acid enantiomers in human plasma by high-performance liquid chromatography

A new method for the simultaneous determination of D- and L-lactic acid in human plasma has been developed using high-performance liquid chromatography (HPLC) with fluorescence detection. This method is based on the reaction of lactic acid with (2S)-2-amino-3-methyl-1-[4-(7-nitro-benzo-2,1,3-oxadiazol-4-yl)-piperazin-1-yl]-butan-1-one (NBD-PZ-Val) in the presence of O-(7-azobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) and N-ethyldiisopropylamine (DIEA) to produce fluorescent diastereomeric derivatives that were easily monitored fluorimetrically at λ(ex)=490 nm and λ(em)=532 nm. The separation was achieved by use of a C18 analytical column (Synergy Hydro 150 mm x 3 mm i.d., 4 μm). The calibration curve was linear over the on-column concentration range of 10-200 μmol/L for D-lactic acid and 0.5-4.0 mmol/L for L-lactic acid. The sensitivity was good with a limit of detection of 5.24 μmol/L for D-lactic acid and 0.15 mmol/L for L-lactic acid. The analytical method was successfully applied to human plasma samples from normal healthy subjects.

Ammonium 5-bromo-7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate: a new fluorogenic reagent for the determination of aminothiols by HPLC.

The measurement of aminothiols such as cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH), as well as the corresponding disulfides, has gained high interest within the biomedical community because such molecules are important biomarkers for a wide range of diseases. In particular, increased total plasma Hcy is now considered a risk factor for cardiovascular disorders(1) as well as other degenerative conditions. Although various methods for the measurement of aminothiols are available, HPLC coupled with fluorometric detection is one of the most suitable techniques for determination of minute amounts of thiols(2). Most methods require precolumn derivatization, and although different types of fluorogenic reagents for thiols have been proposed, the most commonly used and sensitive reagent is the commercially available ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F)(3). With this method the completion of the reaction takes a very long time and the conditions required are quite strict, i.e., derivatization must be carried out at 60 °C for a 1-h period in a moderately alkaline medium (pH = 9.5) in the presence of excess reagent. Moreover, fast …

Identification and structural characterization by LC-ESI-IONTRAP and LC-ESI-TOF of some metabolic conjugation products of homovanillic acid in urine of neuroblastoma patients.

The levels of urinary catecholamine metabolites, such as homovanillic acid (HVA) and vanillylmandelic acid, are routinely used as a clinical tool in the diagnosis and follow-up of neuroblastoma (NB) patients. Recently, in the Clinical Pathology Laboratory Unit of G. Gaslini Children Hospital, a commercial method that employs liquid chromatography coupled to electrochemical detection (LC-EC) has been introduced for the measurement of these metabolites in the routine laboratory practice. Using this LC-EC method, an unknown peak could be observed only in samples derived from NB patients. To investigate the nature of this peak, we used a combination of liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS) and liquid chromatography-ion trap tandem mass spectrometry (LC-IT-MS). The first approach was used to obtain the elemental composition of the ions present in this new signal. To get additional structural information useful for the elucidation of unknown compounds, the ion trap analyzer was exploited. We were able to identify not just one, but three unknown signals in urine samples from NB patients which corresponded to three conjugated products of HVA: HVA sulfate and two glucuronoconjugate isomers. The enzymatic hydrolysis with β-glucuronidase confirmed the proposed structures, while the selective alkaline hydrolysis allowed us to distinguish the difference between phenol- and acyl-glucuronide of HVA. The latter was the unknown peak observed in LC-EC separations of urine samples from NB patients.

Optimised solid phase synthesis of a cystine-linked peptide-PNA chimera

Abstract A solid phase synthesis method for a cystine-linked peptide-PNA chimera starting from different modified protected cystine monomers was developed. This strategy dramatically improved the final yield and the specificity of disulphide bond formation in this kind of oligomers, which are traditionally obtained by liquid phase coupling via oxidation of the two terminal cysteines.