Antonella Aresta

Determination of clenbuterol in human urine and serum by solid-phase microextraction coupled to liquid chromatography.

A solid-phase microextraction (SPME)-LC-UV method for the determination of the beta-adrenergic drug clenbuterol in human urine and serum samples was developed for the first time using a polydimethylsiloxane/divinylbenzene (PDMS/DVB) coated fiber. The procedure required very simple sample pretreatments, isocratic elution, and provided highly selective extractions. All the aspects influencing fiber adsorption (extraction time, temperature, pH, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte have been investigated. The linear ranges investigated in urine and serum were 10-500 and 5-500 ng/ml, respectively (that covers the typical clenbuterol concentration observed in biological fluids). Within-day and between-days R.S.D.% in urine ranged between 5.0-5.3 and 8.5-8.7, respectively, while in serum ranged between 5.5-5.9 and 8.7-9.1, respectively. Estimated LOD and LOQ were 9 and 32 ng/ml (spiked urine), respectively, and 5 and 24 ng/ml (spiked serum), respectively, well below the usual clenbuterol urinary and serum level.

Determination of Ochratoxin A in green coffee beans by solid-phase microextraction and liquid chromatography with fluorescence detection

A new method for the determination of Ochratoxin A (OTA) in green coffee beans by solid-phase microextraction (SPME) coupled to liquid chromatography with fluorescence detection (LC-FD) is described for the first time. Coffee samples were extracted by a 5% NaHCO(3) solution, followed by a clean-up step of the extract by chloroform partition. The aqueous extract was then acidified and finally subjected to SPME-LC-FD analysis. The investigated linear range in coffee was 2-32 ng/g. Within-day RSD% in coffee spiked at 2 and 32 ng/g levels were 3.3 and 2.7, respectively, whereas the between-days RSD% were 4.1 and 3.8, respectively. The limits of detection (LOD) and quantitation (LOQ), calculated at a signal-to-noise ratio of 3 and 10 (noise calculated peak to peak on a blank chromatogram at the OTA retention time), were 0.3 and 2 ng/g, respectively.

Determination of cyclopiazonic acid in cheese samples using solid-phase microextraction and high performance liquid chromatography

Solid phase microextraction (SPME), using a Carbowax/Templated Resin fiber, was optimized for the determination of the mycotoxin cyclopiazonic acid (CPA) and interfaced with HPLC-UV/DAD. All the parameters influencing SPME-HPLC, including extraction time and temperature, pH, ionic strength and desorption conditions, have been carefully examined. The method was successfully applied to the analysis of white surface cheese samples. SPME was capable of a selective extraction of CPA after a short sonication step in methanol. The whole extraction gave high recovery yields and was simpler and quicker than any other existing procedure for CPA extraction from cheese. A detection limit in cheese of 7 ppb was obtained.

Solid-phase microextraction-high performance liquid chromatography and diode array detection for the determination of mycophenolic acid in cheese

Abstract SPME, using a carbowax/templated resin fiber, interfaced with HPLC–UV/DAD has been optimized for the determination of the mycotoxin mycophenolic acid (MPA) in cheese samples. All the parameters influencing the efficiency of the analyte extraction and desorption have been carefully explored. The procedure has been applied to the analysis of blue-cheese samples such as Gorgonzola and Danablu. Samples were subjected to a preliminary short sonication in bicarbonate buffer (0.2 M, pH 9.7); the subsequent SPME was capable of a selective extraction of MPA, characterized by high recovery yields and detection limits of 50 and 100 ppb for Danablu and Gorgonzola, respectively. The present method is faster and simpler than any other existing method for the extraction of MPA from cheese and does not involve the use of toxic organic solvents.

Detection of hazelnut oil in extra-virgin olive oil by analysis of polar components by micro-solid phase extraction based on hydrophilic liquid chromatography and MALDI-ToF mass spectrometry

The oil polar fraction may have a great potential for the characterization of vegetable oils and for the individuation of adulterations. In particular, adulteration of extra-virgin olive oil (EVOO) with hazelnut oil (HO) is one of the most difficult ones to detect due to the similar composition as regards triacylglycerol, total sterol and fatty acid profile. A new micro-solid phase extraction (µ-SPE) procedure based on hydrophilic liquid chromatography (HILIC) micro-columns was developed for the selective extraction and enrichment of polar compounds from EVOO and HO before matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS) analysis. The method permits a simple and fast qualitative analysis of the polar fraction of the oils under study; furthermore, some peaks (such as the m/z ions 496.39, 520.46 and 522.47) were found to be present only in HO, indicating that they could be diagnostic for the presence of HO in EVOO. In order to verify the potential of the method for the individuation of this adulteration, EVOO was progressively adulterated with variable quantities of HO, subjected to the HILIC enrichment and finally to MALDI-ToF-MS analysis; the detection of adulteration was possible up to the level of 5%. Eventually, diagnostic polar compounds were identified as lysophosphatidylcholine (LPC) (16:0/0:0), LPC (18:2/0:0), LPC (18:1/0:0) by means of capillary liquid chromatography-electrospray ionization-quadrupole-ToF-MS (CapLC-ESI-Q-ToF-MS) analysis.

LC-ion trap electrospray MS-MS for the determination of cyclopiazonic acid in milk samples

An LC-ESI-MS (negative ions) method was developed for the determination of cyclopiazonic acid (CPA), a mycotoxin produced by many Aspergillus and Penicillium genuses, in milk samples. First, the acquisition parameters of the ESI mass spectrometer were optimised for deprotonated CPA and MS-MS measurements were performed, giving a fragmentation pattern. After this stage, LC separation was applied to milk extracts (with or without CPA spikes) by means of an aminic column, using the selective reaction monitoring (SRM) acquisition mode for MS detection. The CPA response was linear over three decades of concentration and an LOD of 5 ng mL(-1) was estimated; moreover, the extraction procedure produced almost quantitative recoveries of CPA from milk. Twenty different milk samples were analysed and three of them were found to be contaminated with CPA to various extents.

Transitory DNA hypomethylation during liver cell proliferation induced by a single dose of lead nitrate

In the present study we have examined the effect of a single dose of the mitogen lead nitrate (75 mumols/kg body wt) on the methylation status of hepatic DNA in male Wistar rats. It was found that extensive hypomethylation of hepatic DNA occurs in mitogen-treated rat liver. This effect could be seen as early as 12 h after metal treatment and parallels the changes in liver weight. Probing with the methylation-sensitive enzymes HpaII, MspI, and HaeIII confirmed HPLC analyses and showed that methylation at these sites was affected by lead treatment. DNA hypomethylation has already been found in regenerating rat liver and in hepatic (pre)malignant lesions when compared to normal nondividing liver. Thus the lowering of the DNA 5-methylcytosine content appears to be a property characteristic of cellular proliferation, regardless of whether it is caused by partial hepatectomy, carcinogen treatments, or mitogen administration.

Determination of delorazepam in urine by solid-phase microextraction coupled to high performance liquid chromatography

An SPME-HPLC-UV method for the determination of delorazepam, a representative benzodiazepine, in spiked human urine samples was developed for the first time. The performances of two commercially available fibers, a carbowax/templated resin (Carbowax/TPR-100) and a polydimethylsiloxane/divinylbenzene (PDMS/DVB), were compared, indicating the latter as the most suitable for urine samples analysis. All the aspects influencing adsorption (extraction time, pH, temperature, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte on the fiber have been investigated. In particular, short extraction times were necessary to reach the equilibrium and very short desorption times were employed. The procedure required simple sample pre-treatment and was able to detect 5 ng/ml in spiked urine, regardless of the complexity of the matrix.

SPME-LC with UV detection to study delorazepam -serum albumin interactions

Solid phase microextraction coupled to high performance liquid chromatography with UV detection (SPME/LC-UV) has been employed to study the binding of delorazepam to human serum albumin (HSA) and bovine serum albumin (BSA). The procedure could also be potentially extended to the measurement of partition coefficients between a wide variety of semi- or non-volatile compounds and matrices. The method is solvent free, simple, fast, and drawbacks of the conventional analytical techniques are avoided. Moreover, the matrix did not interfere with the measurement by binding to the fibre and the amount extracted by the fibre was negligibly small; thus it did not disturb the delorazepam-protein binding.

Amino-bonded silica as stationary phase for liquid chromatographic determination of cyclopiazonic acid in fungal extracts.

A new high-performance liquid (HPLC) chromatographic method is described for cyclopiazonic acid (CPA) determination in fungal cultures on a propylamino-bonded stationary phase with a CH3CN/CH3COONH4 buffer as mobile phase. Retention of CPA on propylamino modified silica under acidic conditions (protonated amino groups and deprotonated CPA) is governed by a mixed ion-exchange-reversed-phase mechanism. In addition to non-polar (hydrophobic) interactions, polar interactions with the surface silanols are also possible and become important as the polarity of the mobile phase decreases. A detection limit of 25 pg of CPA standard is obtained that represents an improvement of more than two orders of magnitude compared to existing HPLC procedures. UV-detector response was linear to 200 ng of CPA. Fungal extracts can be analysed after a simple dilution step with UV diode array detection that provides peak identity/purity assessment. The suitability of the proposed method as a rapid confirmatory test to assess the toxigenic potential of different Aspergillus and Penicillium strains is demonstrated by the analysis of 54 fungal extracts.