Luigi Silvestro

A Confirmatory HPLC–MS/MS Method for Ten Synthetic Corticosteroids in Bovine Urines

In the present study, an HPLC-MS/MS method to confirm, in bovine urine, the most common synthetic corticosteroids illegally used as growth promoters in livestock breeding will be presented. An API III-Plus (PE-Sciex) triple quadrupole mass spectrometer, interfaced by means of an atmospheric pressure chemical ionization source to the HPLC system, was used. Urine samples were treated with a sulfatase-glucuronidase mixture to cleave the drug-conjugates and then extracted on C18 disposable columns. LC separations were performed on a reversed-phase C18 column with ammonium acetate 0.1 M/acetonitrile (60/40, v/v) as mobile phase. Detection was performed in multiple reaction monitoring mode, negative ions, selecting fragmentations characteristic of 10 corticosteroids used more frequently. Good results, in terms of sensitivity and specificity have been obtained for nine corticosteroids that can be analyzed in the same HPLC run; the limits of sensitivity achieved were 0.05-1.0 ng/ml in urine. Only a more polar corticosteroid, required a different HPLC separation. Practical applications of this technique to real samples proved that it is an effective method to confirm the illegal use of corticosteroids as growth promoter in animal. In comparison with the chemical GC-MS methods the simpler sample preparation and the faster time of analysis permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity.

Potential angiogenic role of platelet-activating factor in human breast cancer.

This study investigated the presence of platelet-activating factor (PAF) in the lipid extracts of 18 primary breast carcinomas and 20 control breast tissues. The amount of PAF detected in breast carcinomas was significantly higher than in controls. The mass spectrometric analysis of PAF-bioactive lipid extract from breast carcinomas showed the presence of several molecular species of PAF, including C16-alkylPAF, C18-lysophosphatidylcholine (LPC), C16-LPC, lyso-PAF, and C16-acylPAF. The amount of bioactive PAF extracted from breast specimens significantly correlated with tumor vascularization revealed by the number of CD34-and CD31-positive cells. As C16-alkylPAF was previously shown to induce angiogenesis in vivo, we evaluated whether the thin layer chromatography-purified lipid extracts of breast specimens elicited neoangiogenesis in a murine model of subcutaneous Matrigel injection. The lipid extracts from specimens of breast carcinoma containing high levels of PAF bioactivity, but not from breast carcinomas containing low levels of PAF bioactivity or from normal breast tissue, induced a significant angiogenic response. This angiogenic response was significantly inhibited by the PAF receptor antagonist WEB 2170. T47D and MCF7 breast cancer cell lines, but not an immortalized nontumor breast cell line (MCF10), released PAF in the culture medium. A significant in vivo neoangiogenic response, inhibited by WEB 2170, was elicited by T47D and MCF7 but not by MCF10 culture medium. These results indicate that an increased concentration of PAF is present in tumors with high microvessel density and that PAF may account for the neoangiogenic activity induced in mice by the lipid extracts obtained from breast cancer. A contribution of PAF in the neovascularization of human breast cancer is suggested.

Plasmin Promotes an Endothelium-Dependent Adhesion of Neutrophils Involvement of Platelet Activating Factor and P-Selectin

BACKGROUND The adhesion of leukocytes to the endothelium and the edema of vessel wall may cause vascular reocclusion after thrombolytic therapy. The aim of this study was to evaluate the role of platelet activating factor (PAF) and P-selectin on the adherence of polymorphonuclear neutrophils (PMN) to the endothelium and of PAF on the increased vascular permeability induced by tissue-type plasminogen activator, streptokinase, and plasmin. METHODS AND RESULTS We studied (1) the adhesion of 111Inlabeled PMN to human umbilical cord vein-derived cultured endothelial cells (HUVEC), (2) the transfer of 125I-labeled albumin across HUVEC monolayers, and (3) the adhesion of PMN to isolated bovine coronary arteries under flow conditions. It was found that the adhesion of PMN, induced by tissue-type plasminogen activator, streptokinase, and plasmin, correlated with the synthesis of PAF by HUVEC and was inhibited by WEB 2170, a PAF receptor antagonist. The adhesion of PMN was also inhibited by the treatment of HUVEC with anti-P-selectin antibodies or of PMN with soluble P-selectin or with anti-CD18 monoclonal antibodies. Plasmin also increased the permeability of HUVEC monolayers, an effect that was partially prevented by WEB 2170. Moreover, plasmin promoted the synthesis of PAF from isolated bovine coronary arteries and the adherence of PMN to the endothelium under flow conditions. The pretreatment of PMN with WEB 2170 or with soluble P-selectin prevented adhesion. CONCLUSIONS The synthesis of PAF by endothelial cells at the site of plasmin generation and the endothelial expression of P-selectin may render the endothelial cell surface proadhesive for neutrophils and may favor a local increase in vascular permeability.

Development and validation of an HPLC–MS/MS method to determine clopidogrel in human plasma. Use of incurred samples to test back-conversion

Quantitative methods using LC-MS/MS allow achievement of adequate sensitivity for pharmacokinetic studies with clopidogrel; three such methods, with LLOQs as low as 5 pg/mL, were developed and fully validated according to the well established FDA 2001 guidelines. The chromatographic separations were performed on reversed phase columns Ascentis RP-Amide (15 cm x 2.1 mm, 5 μm), Ascentis Express C8 (10 cm x 2.1 mm, 2.7 μm) and Ascentis Express RP Amide (10 cm x 2.1 mm, 2.7 μm), respectively. Positive electrospray ionization in MRM mode was employed for the detection and a deuterated analogue (d3-clopidogrel) was used as internal standard. Linearity, precision, extraction recovery, matrix effects and stability tests on blank plasma spiked with clopidogrel and stored in different conditions met the acceptance criteria. During the analysis of the real samples from the first pharmacokinetic study, a significant increase (>100%) of the measured clopidogrel concentrations in the extracts kept in the autosampler at 10 °C was observed. Investigations led to the conclusion that most probably a back-conversion of one or more of the clopidogrel metabolites is occurring. The next methods were optimized in order to minimize this back-conversion. After a series of experiments, the adjustment of the sample preparation (e.g. processing at low temperature and introducing a clean-up step on Supelco HybridSPE-Precipitation cartridges) has proven to be the most effective in order to improve the stability of the extracts. Incurred samples of real subjects were successfully used in the validation of the last two analytical methods to evaluate the back-conversion, while tests using only the known metabolites could not detect this important problem.

Characterization of the chemical structure of sulphated glycosaminoglycans after enzymatic digestion. Application of liquid chromatography-mass spectrometry with an atmospheric pressure interface.

Pneumatically assisted electrospray was demonstrated to be a powerful ionization source for the analysis of oligosaccharides. A mass spectrometer was interfaced to an HPLC system, using this interface, to determine oligosaccharides from the enzymatic digestion of heparin separated on a reversed-phase column. To set up the technique, and particularly to clarify the ionization process, purified disaccharides, from enzymatic digestion of chondroitin sulphates, were measured. The use of a suitable counter ion in the mobile phase, tetrapropylammonium (TPA), to optimize the HPLC separation, gave, with sulphated di- and oligosaccharides, adducts [M + nTPA - (n + m)H]m-, which were unexpectedly stable to fragmentation; molecular ions [M - (n + 1)H]n-, in the presence of the counter ion, were observed only with desulphated or monosulphated disaccharides. The stability of the adducts and the use of a deuterated ion-pair reagent permitted an exact evaluation of the molecular masses of disaccharides and oligosaccharides of unknown structure. Spectra obtained in the absence of the counter ion contained singly or multiply charged molecular ions and fragmentation ions mainly from loss of the sulphate groups; under these ionization conditions the exact mass determination and interpretation of the spectra were difficult. After removal of the counter ion, tandem mass spectra could be obtained with some interesting data for the characterization of these molecules. Complete spectral analyses were performed with amounts of samples of 50 micrograms but, using microbore columns, one twentieth of this amount may give good spectra.

Development of a high-performance liquid chromatographic-mass spectrometric technique, with an ionspray interface, for the determination of platelet-activating factor (PAF) and lyso-PAF in biological samples.

An HPLC-mass spectrometric technique with an ionspray interface was developed for the determination of platelet-activating factor (PAF) and PAF-related compounds in biological samples. HPLC separations were performed using a reversed-phase column. The mass spectra showed intense [M + H]+ ions. Collision-induced dissociation of protonated molecular ions gave characteristic daughter ions corresponding to the phosphorylcholine group. By selective-ion monitoring, a detection limit of 0.3 ng was obtained for all molecules; by multiple reaction monitoring, the same sensitivity was achieved for PAF whereas for lyso-PAF the limit was 3 ng. Finally, PAF was comparatively determined by bioassay and HPLC-MS after extraction from the cell pellets and the supernatants of human polymorphonuclear neutrophils unstimulated or stimulated with opsonized zymosan. The good correlation observed between these techniques indicated the reliability of HPLC-MS for biochemical studies on PAF and PAF-related molecules.

High-performance liquid chromatographic—mass spectrometric analysis of cis-dichlorodiamineplatinum—DNA complexes using an ionspray interface

Abstract An efficient RP-HPLC separation technique was used in combination with mass spectrometric detection with an ionspray ionization source to analyse complexes between nucleosides and cis -dichlorodiamineplatinum(II). Conventional detection techniques (UV and atomic absorption spectrometry) were also used as starting points for the setting-up of this HPLC-MS approach. The method was developed using complexes obtained either by reaction of free deoxynucleosides with cis -dichlorodiamineplatinum or by reaction in vitro of DNA samples with the same drug. DNA samples before HPLC-MS were completely depolymerized by digestion with nuclease P1 and alkaline phosphatase, in order easily to separate and determine the complexes formed. The sensitivity obtained makes this technique very suitable for future application in biological studies. The detection level, defined as the detector response with a signal-to-noise ratio of 2, corresponds to 2 pmol injected. In DNA samples treated with cis -dichlorodiamineplatinum, a series of cis -dichlorodiamineplatinum—deoxynucleoside complexes not previously described were also detected.

High-performance liquid chromatographic-mass spectrometric analysis of oligosaccharides from enzymatic digestion of glycosaminoglycans: Application to human samples☆

Glycosaminoglycan contents were evaluated in plasma and urine samples from volunteers treated intravenously with a mixture of dermatan sulphate and heparin, combining a novel liquid chromatographic-mass spectrometric technique for the determination of oligosaccharides from glycosaminoglycans with a classical technique for the extraction of glycosaminoglycans from biological samples (precipitation with cetylpyridinium chloride). In plasma samples dermatan sulphate and heparin can be measured for 2 h after treatment; urine excretion was detectable for 24 h. These results suggest that this novel approach is promising for future studies on the pharmacokinetics of glycosaminoglycans, although some technical aspects need further improvement, mainly regarding the procedures for sample clean-up; cetylpiridinium precipitation is a complex procedure and the recovery is limited.

High-throughput HPLC-MS/MS method to determine ibandronate in human plasma for pharmacokinetic applications

A sensitive high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of ibandronate in human plasma. In a previous study, we have analyzed alendronate in urine samples of subjects treated at therapeutic dosages, using a derivatization approach; a similar derivatization was adapted and improved to determine ibandronate in plasma. The bisphosphonate was isolated from the biological matrix by liquid-liquid extraction, and derivatized with trimethylsilyldiazomethane prior to separation on a reversed-phase column (Supelco Discovery HSC18) and detection on a quadrupole-linear ion trap mass spectrometer (API 4000 QTrap). Various parameters of extraction and derivatization were optimized in order to get adequate recovery, high derivatization yield and minimal ion suppression; a deuterated analogue, d3-ibandronate, was used as internal standard. The transitions 376.1-->114.2 and 379.1-->61.0 were acquired to monitor ibandronate and d3-ibandronate derivatives, respectively. A multiplexing LC system made possible the overlapping of two chromatographic runs, thus the interval between injections being reduced to only 2min, a very short analysis time for compounds of this class. The method was fully validated over the quantification range 0.2-175.0ng/ml, allowing an appropriate evaluation of the plasma concentrations of ibandronate, expected at therapeutic dosage, as proved by application to a pharmacokinetic study. A good linearity over the selected range (r>0.99), accuracy and precision within +/-15% of the target values and a recovery over 50% were obtained.

Determination of 1-O-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, platelet-activating factor and related phospholipids in biological samples by high-performance liquid chromatography-tandem mass spectrometry

Combining normal-phase HPLC separation and tandem mass spectrometric detection, using an ion-spray HPLC-MS interface, a quantitative method for acyl-platelet activating factor (acyl-PAF), platelet-activating factor (PAF) and related phospholipids was developed. Mass spectra, positive ions, showed intense [M+H]+ ions; collision-induced dissociation of protonated molecular ions gave characteristic daughter ions corresponding to the polar head. Detection limits of 0.1-0.3 ng injected were obtained by multiple reaction monitoring. Samples of human endothelial cells treated with compounds modulating the levels of acyl-PAF and PAF have been analyzed by the present technique, proving that this approach is suitable for biochemical studies.