Discovery of Clinical Candidate CHF-6366: A Novel Super-soft Dual Pharmacology Muscarinic Antagonist and ß2 Agonist (MABA) for the Inhaled Treatment of Respiratory Diseases.

The development of molecules embedding two distinct pharmacophores acting as muscarinic antagonists and ß2 agonists (MABAs) promises to be an excellent opportunity to reduce formulation issues and boost efficacy through cross-talk and allosteric interactions. Herein, we report the results of our drug discovery campaign aimed at improving the therapeutic index of a previous MABA series by exploiting the super soft-drug concept. The incorporation of a metabolic liability, stable at the site of administration but undergoing rapid systemic metabolism, to generate poorly active and quickly eliminated fragments was pursued. Our SAR studies yielded MABA 29, which demonstrated a balanced in vivo profile up to 24 h, high instability in plasma and the liver, as well as sustained exposure in the lung. In vitro safety and non-GLP toxicity studies supported the nomination of 29 (CHF-6366) as a clinical candidate, attesting to the successful development of a novel super-soft MABA compound.

Pulmonary Surfactant: A Unique Biomaterial with Life-saving Therapeutic Applications.

Pulmonary surfactant is a complex lipoprotein mixture secreted into the alveolar lumen by type 2 pneumocytes, which is composed by tens of different lipids (approximately 90% of its entire mass) and surfactant proteins (approximately 10% of the mass). It is crucially involved in maintaining lung homeostasis by reducing the values of alveolar liquid surface tension close to zero at end-expiration, thereby avoiding the alveolar collapse, and assembling a chemical and physical barrier against inhaled pathogens. A deficient amount of surfactant or its functional inactivation is directly linked to a wide range of lung pathologies, including the neonatal respiratory distress syndrome. This paper reviews the main biophysical concepts of surfactant activity and its inactivation mechanisms, and describes the past, present and future roles of surfactant replacement therapy, focusing on the exogenous surfactant preparations marketed worldwide and new formulations under development. The closing section describes the pulmonary surfactant in the context of drug delivery. Thanks to its peculiar composition, biocompatibility, and alveolar spreading capability, the surfactant may work not only as a shuttle to the branched anatomy of the lung for other drugs but also as a modulator for their release, leading to innovative therapeutic avenues for the treatment of several respiratory diseases.

In vitro characterization and in vivo comparison of the pulmonary outcomes of Poractant alfa and Calsurf in ventilated preterm rabbits.

Poractant alfa and Calsurf are two natural surfactants widely used in China for the treatment of neonatal respiratory distress syndrome, which are extracted from porcine and calf lungs, respectively. The purpose of this experimental study was to compare their in vitro characteristics and in vivo effects in the improvement of pulmonary function and protection of lung injury. The biophysical properties, ultrastructure, and lipid composition of both surfactant preparations were respectively analysed in vitro by means of Langmuir-Blodgett trough (LBT), atomic force microscopy (AFM), and liquid-chromatography mass-spectrometry (LC-MS). Then, as core pharmacological activity, both head-to-head (100 and 200 mg/kg for both surfactants) and licensed dose comparisons (70 mg/kg Calsurf vs. 200 mg/kg Poractant alfa) between the two surfactants were conducted as prophylaxis in preterm rabbits with primary surfactant deficiency, assessing survival time and rate and dynamic compliance of the respiratory system (Cdyn). Intrapulmonary surfactant pools, morphometric volume density as alveolar expansion (Vv), and lung injury scores were determined post mortem. AFM and LC-MS analysis revealed qualitative differences in the ultrastructure as well as in the lipid composition of both preparations. Calsurf showed a longer plateau region of the LBT isotherm and lower film compressibility. In vivo, both surfactant preparations improved Cdyn at any dose, although maximum benefits in terms of Vv and intrapulmonary surfactant pools were seen with the 200 mg/kg dose in both surfactants. The group of animals treated with 200 mg/kg of Poractant alfa showed a prolonged survival time and rate compared to untreated but ventilated controls, and significantly ameliorated lung injury compared to Calsurf at any dose, including 200 mg/kg. The overall outcomes suggest the pulmonary effects to be dose dependent for both preparations. The group of animals treated with 200 mg/kg of Poractant alfa showed a significant reduction of mortality. Compared to Calsurf, Poractant alfa exerted better effects if licensed doses were compared, which requires further investigation.

Quenching of tryptophan fluorescence in a highly scattering solution: Insights on protein localization in a lung surfactant formulation.

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic surfactant developed for respiratory distress syndrome replacement therapy in pre-term newborn infants. CHF5633 contains two phospholipids (dipalmitoylphosphatidylcholine and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol sodium salt), and peptide analogues of surfactant protein C (SP-C analogue) and surfactant protein B (SP-B analogue). Both proteins are fundamental for an optimal surfactant activity in vivo and SP-B genetic deficiency causes lethal respiratory failure after birth. Fluorescence emission of the only tryptophan residue present in SP-B analogue (SP-C analogue has none) could in principle be exploited to probe SP-B analogue conformation, localization and interaction with other components of the pharmaceutical formulation. However, the high light scattering activity of the multi-lamellar vesicles suspension characterizing the pharmaceutical surfactant formulation represents a challenge for such studies. We show here that quenching of tryptophan fluorescence and Singular Value Decomposition analysis can be used to accurately calculate and subtract background scattering. The results indicate, with respect to Trp microenvironment, a conformationally homogeneous population of SP-B. Trp is highly accessible to the water phase, suggesting a surficial localization on the membrane of phospholipid vesicles, similarly to what observed for full length SP-B in natural lung surfactant, and supporting an analogous role in protein anchoring to the lipid phase.

Discovery and Optimization of Thiazolidinyl and Pyrrolidinyl Derivatives as Inhaled PDE4 Inhibitors for Respiratory Diseases.

Phosphodiesterase 4 (PDE4) is a key cAMP-metabolizing enzyme involved in the pathogenesis of inflammatory disease, and its pharmacological inhibition has been shown to exert therapeutic efficacy in chronic obstructive pulmonary disease (COPD). Herein, we describe a drug discovery program aiming at the identification of novel classes of potent PDE4 inhibitors suitable for pulmonary administration. Starting from a previous series of benzoic acid esters, we explored the chemical space in the solvent-exposed region of the enzyme catalytic binding pocket. Extensive structural modifications led to the discovery of a number of heterocycloalkyl esters as potent in vitro PDE4 inhibitors. (S*,S**)-18e and (S*,S**)-22e, in particular, exhibited optimal in vitro ADME and pharmacokinetics properties and dose-dependently counteracted acute lung eosinophilia in an experimental animal model. The optimal biological profile as well as the excellent solid-state properties suggest that both compounds have the potential to be effective topical agents for treating respiratory inflammatory diseases.

In vitro and in vivo characterization of poractant alfa supplemented with budesonide for safe and effective intratracheal administration.

BackgroundThe intratracheal (IT) administration of budesonide using surfactant as a vehicle has been shown to reduce the incidence of bronchopulmonary dysplasia (BPD) in preterm infants. The objective of this study was to characterize the in vitro characteristics and in vivo safety and efficacy of the extemporaneous combination of budesonide and poractant alfa.MethodsThe stability, minimum surface tension, and viscosity of the preparation were evaluated by means of high-performance liquid chromatography (HPLC), Wilhelmy balance, and Rheometer, respectively. The safety and efficacy of the IT administration of the mixture were tested in two respiratory distress syndrome (RDS) animal models: twenty-seventh day gestational age premature rabbits and surfactant-depleted adult rabbits.ResultsA pre-formulation trial identified a suitable procedure to ensure the homogeneity and stability of the formulation. Wilhelmy Balance tests clarified that budesonide supplementation has no detrimental effect on poractant alfa surface tension activity. The addition of budesonide to poractant alfa did not affect the physiological response to surfactant treatment in both RDS animal models, and was associated to a significant reduction of lung inflammation in surfactant-depleted rabbits.ConclusionOur in vitro and in vivo analysis suggests that the IT administration of a characterized extemporaneous combination of poractant alfa and budesonide is a safe and efficacious procedure in the context of RDS.

Physiological, Biochemical, and Biophysical Characterization of the Lung-Lavaged Spontaneously-Breathing Rabbit as a Model for Respiratory Distress Syndrome.

Nasal continuous positive airway pressure (nCPAP) is a widely accepted technique of non-invasive respiratory support in spontaneously-breathing premature infants with respiratory distress syndrome (RDS). Surfactant administration techniques compatible with nCPAP ventilation strategy are actively investigated. Our aim is to set up and validate a respiratory distress animal model that can be managed on nCPAP suitable for surfactant administration techniques studies. Surfactant depletion was induced by bronchoalveolar lavages (BALs) on 18 adult rabbits. Full depletion was assessed by surfactant component analysis on the BALs samples. Animals were randomized into two groups: Control group (nCPAP only) and InSurE group, consisting of a bolus of surfactant (Poractant alfa, 200 mg/kg) followed by nCPAP. Arterial blood gases were monitored until animal sacrifice, 3 hours post treatment. Lung mechanics were evaluated just before and after BALs, at the time of treatment, and at the end of the procedure. Surfactant phospholipids and protein analysis as well as surface tension measurements on sequential BALs confirmed the efficacy of the surfactant depletion procedure. The InSurE group showed a significant improvement of blood oxygenation and lung mechanics. On the contrary, no signs of recovery were appreciated in animals treated with just nCPAP. The surfactant-depleted adult rabbit RDS model proved to be a valuable and efficient preclinical tool for mimicking the clinical scenario of preterm infants affected by mild/moderate RDS who spontaneously breathe and do not require mechanical ventilation. This population is of particular interest as potential target for the non-invasive administration of surfactant.

CHF6001 II: a novel phosphodiesterase 4 inhibitor, suitable for topical pulmonary administration--in vivo preclinical pharmacology profile defines a potent anti-inflammatory compound with a wide therapeutic window.

CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide] is a novel phosphodiesterase 4 (PDE4) inhibitor designed for use in pulmonary diseases by inhaled administration. Intratracheal administration of CHF6001 to ovalbumin-sensitized Brown-Norway rats suppressed the antigen-induced decline of lung functions (ED50 = 0.1 µmol/kg) and antigen-induced eosinophilia (ED50 = 0.03 µmol/kg) when administered (0.09 µmol/kg) up to 24 hours before antigen challenge, in agreement with CHF6001-sustained lung concentrations up to 72 hours after intratracheal treatment (mean residence time 26 hours). Intranasal, once daily administration of CHF6001 inhibited neutrophil infiltration observed after 11 days of tobacco smoke exposure in mice, both upon prophylactic (0.15-0.45 µmol/kg per day) or interventional (0.045-0.45 µmol/kg per day) treatment. CHF6001 was ineffective in reversing ketamine/xylazine-induced anesthesia (a surrogate of emesis in rat) up to 5 µmol/kg administered intratracheally, a dose 50- to 150-fold higher than anti-inflammatory ED50 observed in rats. When given topically to ferrets, no emesis and nausea were evident up to 10 to 20 µmol/kg, respectively, whereas the PDE4 inhibitor GSK-256066 (6-[3-(dimethylcarbamoyl)phenyl]sulfonyl-4-(3-methoxyanilino)-8-methylquinoline-3-carboxamide) induced nausea at 1 µmol/kg intratracheally. A 14-day inhalation toxicology study in rats showed a no-observed-adverse-effect level dose of 4.4 µmol/kg per day for CHF6001, lower than the 0.015 µmol/kg per day for GSK-256066. CHF6001 was found effective and extremely well tolerated upon topical administration in relevant animal models, and may represent a step forward in PDE4 inhibition for the treatment of asthma and chronic obstructive respiratory disease.

Surfactant protein C metabolism in human infants and adult patients by stable isotope tracer and mass spectrometry.

Surfactant protein C (SP-C) is deemed as the surfactant protein most specifically expressed in type II alveolar epithelial cells and plays an important role in surfactant function. SP-C turnover in humans and its meaning in the clinical context have never been approached. In this study, we used mass spectrometry to investigate SP-C turnover in humans. We studied four infants and eight adults requiring mechanical ventilation. All patients had no lung disease. Patients received a 24-h continuous infusion of (13)C-leucine as precursor of SP-C, and serial tracheal aspirates and plasma samples were obtained every 6 h till 48 h. SP-C was isolated from tracheal aspirates by sorbent-phase chromatography. (13)C-leucine SP-C enrichment could be successfully measured in three infant and in four adult samples by using mass spectrometry coupled with a gas chromatographer. Median SP-C fractional synthesis rate, secretion time, and peak time were 15.7 (14.1-27.5)%/day, 6.0 (4.7-11.5) h, and 24 (20-27) h. In conclusion, this study shows that it is feasible to accurately determine SP-C turnover in humans by stable isotopes.

Novel class of benzoic acid ester derivatives as potent PDE4 inhibitors for inhaled administration in the treatment of respiratory diseases.

The first steps in the selection process of a new anti-inflammatory drug for the inhaled treatment of asthma and chronic obstructive pulmonary disease are herein described. A series of novel ester derivatives of 1-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3,5-dichloropyridin-4-yl) ethanol have been synthesized and evaluated for inhibitory activity toward cAMP-specific phosphodiesterase-4 (PDE4). In particular, esters of variously substituted benzoic acids were extensively explored, and structural modification of the alcoholic and benzoic moieties were performed to maximize the inhibitory potency. Several compounds with high activity in cell-free and cell-based assays were obtained. Through the evaluation of opportune in vitro ADME properties, a potential candidate suitable for inhaled administration in respiratory diseases was identified and tested in an in vivo model of pulmonary inflammation, proving its efficacy.

Impact of drug administration route on drug delivery and distribution into the lung: an imaging mass spectrometry approach.

During the last decade, significant technological improvements in mass spectrometry have had a great impact on drug discovery. The development of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has set a new frontier for the study of the distribution of endogenous and exogenous molecules present within a tissue. MALDI-IMS is a surface sampling technique that allows not only the detection of multiple analytes but also gives the spatial distribution of those analytes. Active compounds for pulmonary disease need an optimal and well-studied delivery into the lungs, in order to assure distribution with greater penetration into the peripheral or the alveolar region of the lung to maximize the therapeutic effects. IMS is very useful in the field of drug discovery, showing drug delivery and distribution in the body and organs. In this study, we present a comparison between two different ways of carrying out pulmonary drug administration: inhalation of a nebulized aerosol of aqueous drug solutions and intratracheal administration, which is much simpler, not expensive and commonly used during in vivo screening. Tiotropium bromide is a long-acting anticholinergic medicine used for maintenance treatment of chronic obstructive pulmonary disease. In the present work, tiotropium was administered by nebulization and by intratracheal instillation to guinea pigs at doses able to induce significant anti-bronchoconstrictive activity. Lung samples were dissected, frozen, cryosectioned and coated with matrix (α-hydroxy-cinnamic acid). IMS analyses were performed using a MALDI-LTQ-Orbitrap XL. Using this technique we were able to compare different distributions of the drug depending on the method of administration.

Characterization of the phosphoproteome in human bronchoalveolar lavage fluid.

Global-scale examination of protein phosphorylation in human biological fluids by phosphoproteomics approaches is an emerging area of research with potential for significant contributions towards discovery of novel biomarkers. In this pilot work, we analyzed the phosphoproteome in human bronchoalveolar lavage fluid (BAL) from nondiseased subjects. The main objectives were to assess the feasibility to probe phosphorylated proteins in human BAL and to obtain the initial catalog of BAL phosphoproteins, including protein identities and exact description of their phosphorylation sites. We used a gel-free bioanalytical workflow that included whole-proteome digestion of depleted BAL proteins, enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC), LC-MS/MS analyses with a linear ion trap mass spectrometer, and searches of a protein sequence database to generate a panel of BAL phosphoproteins and their sites of phosphorylation. Based on sequence-diagnostic MS/MS fragmentation patterns, we identified a collection of 36 phosphopeptides that contained 26 different phosphorylation sites. These phosphopeptides mapped to 21 phosphoproteins including, for example, vimentin, plastin-2, ferritin heavy chain, kininogen-1, and others. The characterized phosphoproteins have diverse characteristics in terms of cellular origin and biological function. To the best of our knowledge, results of this study represent the first description of the human BAL phosphoproteome.

Evaluation of lung inflammation induced by intratracheal administration of LPS in mice: comparison between MRI and histology.

PURPOSE: To study the in vivo effect of intratracheal administration of lipopolysaccharide (LPS) in mice by magnetic resonance imaging (MRI) and to investigate the correlation with ex vivo histological evaluation of lung inflammation and oedema. MATERIALS AND METHODS: LPS (or phosphate buffered saline) was administered intratracheally to thirty male Balb/C mice at a concentration of 0.3 mg/ml in a total volume of 100 microl. Animals were divided into fifteen LPS-treated and fifteen control mice. MR images were acquired 24 h after challenge in freely breathing animals with standard ECG-gated Gradient-Echo (GRE) sequences and, in a limited number of animals, with ECG-gated Ultrashort-echo time (UTE) sequences. After MRI, animals were sacrificed, and lungs were fixed and processed for histological analysis of the total volume of healthy lung tissue. RESULTS: GRE images revealed the presence of high intensity signal in lungs of LPS-treated mice that was attributable to oedema caused by alveolar inflammation. In histological slices, regions of alterations in the normal alveolar microstructure were observed that could account for MRI findings. A good correlation was observed between the volumes of lesioned tissue measured by MRI and by histology. The volume of the lesion detected by GRE sequences was lower than the volume detected by UTE sequences. CONCLUSIONS: The effect of intratracheal administration of LPS in mice was investigated by MRI and histology. A good correlation was observed between GRE-MRI and histological findings. MR images obtained with UTE sequences appear to be more sensitive to the presence of lesions than those obtained by standard GRE acquisitions.

1H NMR discrimination of CHF4226.01 diastereoisomers in DMSO-d6.

Two diastereoisomers CHF4226.01 (R, R) and CHF4232.01 (S, R), differing for a chiral center, have been studied to investigate their possible discrimination using NMR. 1D NMR and 2D NMR experiments, such as COSY, NOESY and ROESY, were performed on pure isomers and on the association complexes formed in the presence of the chiral reagent (S)-(-)-1-(2-napthyl)ethylamine (S-NEA). Moreover, computational studies, concerning conformational analysis and molecular dynamics, were started and supported the NMR results.

Alpha,beta-unsaturated aldehydes in cigarette smoke release inflammatory mediators from human macrophages.

Smoking cigarettes is the major risk factor for chronic obstructive pulmonary disease (COPD). COPD is a condition associated with chronic pulmonary inflammation, characterized by macrophage activation, neutrophil recruitment, and cell injury. Many substances contained in cigarette smoke, including reactive oxygen species (ROS), have been proposed to be responsible for the inflammatory process of COPD. However, this issue remains unsettled. By gas chromatography/mass spectrometry (GC/MS) we show that acrolein and crotonaldehyde, two alpha,beta-unsaturated aldehydes, are contained in aqueous cigarette smoke extract (CSE) at micromolar concentrations and mimic CSE in evoking the release of the neutrophil chemoattractant IL-8 and of the pleiotropic inflammatory cytokine TNF-alpha from the human macrophagic cell line U937. In addition, acrolein (10-30 microM) released IL-8 also from cultured human alveolar macrophages and THP-1 macrophagic cells. 4-hydroxy-2-nonenal (30-100 microM), an endogenous alpha,beta-unsaturated aldehyde that is abundant in lungs of patients with COPD, stimulated the release of IL-8 from U937 cells, whereas the saturated aldehyde, acetaldehyde, was ineffective. CSE-evoked IL-8 release was remarkably (> 80%) inhibited by N-acetyl-cysteine (0.1-3 mM) or glutathione monoethyl ester (1-3 mM). Both compounds, by forming covalent adducts (Michael adducts), completely removed unsaturated aldehydes from CSE. Our data demonstrate that alpha,beta-unsaturated aldehydes are major mediators of cigarette smoke-induced macrophage activation, and suggest that they might contribute to pulmonary inflammation associated with cigarette smoke.

Synthesis and biological activity of flurbiprofen analogues as selective inhibitors of beta-amyloid(1)(-)(42) secretion.

Flurbiprofen, a nonsteroidal antiinflammatory drug (NSAID), has been recently described to selectively inhibit beta-amyloid(1)(-)(42) (Abeta42) secretion, the most toxic component of the senile plaques present in the brain of Alzheimer patients. The use of this NSAID in Alzheimer's disease (AD) is hampered by a significant gastrointestinal toxicity associated with cyclooxygenase (COX) inhibition. New flurbiprofen analogues were synthesized, with the aim of increasing Abeta42 inhibitory potency while removing anti-COX activity. In vitro ADME developability parameters were taken into account in order to identify optimized compounds at an early stage of the project. Appropriate substitution patterns at the alpha position of flurbiprofen allowed for the complete removal of anti-COX activity, while modifications at the terminal phenyl ring resulted in increased inhibitory potency on Abeta42 secretion. In rats, some of the compounds appeared to be well absorbed after oral administration and to penetrate into the central nervous system. Studies in a transgenic mice model of AD showed that selected compounds significantly decreased plasma Abeta42 concentrations. These new flurbiprofen analogues represent potential drug candidates to be developed for the treatment of AD.

High throughput screening of beta-amyloid secretion inhibitors using homogenous time-resolved fluorescence.

A cell-based assay using homogeneous time-resolved fluorescence has been developed for high throughput screening of putative beta-amyloid (Abeta)production inhibitors. In this assay, total Abeta is detected by simply adding two commercially available antibody complexes. The first was a biotinylated monoclonal antibody (4G8), specifically recognizing an epitope comprising the residues 17-24 of the Abetapeptide, complexed with europium cryptate-streptavidin conjugate. The second was a polyclonal antibody (BioS-N), raised against the N-terminus of the Abeta peptide, complexed with an allophycocyanin-anti rabbit antibody conjugate. Binding of the two complexes to the Abeta peptide brought europium cryptate (fluorescence donor) and allophycocyanin (fluorescence acceptor) into close proximity, consequently a fluorescent resonance energy transfer signal was produced upon excitation at 337 nm. The resulting fluorescence signal (665 nm) was then detected using a Discovery or a ViewLux reader. Detection of Abeta by the proposed method is possible at concentrations of approximately 1 nM. The method was employed for the detection of Abeta secreted from a stable transfected human neuroglioma cell line (H4) overexpressing a mutated form of the human amyloid precursor protein (APP695NL) and developed for robotic automation. At optimized conditions, signal-to-background ratios exceeding 5 and Z' factors around 0.7 were achieved in a 384-well format. High throughput screening of 56,913 potential Abeta production inhibitors led to identification of new non-cytotoxic and cell permeable compounds with potencies in the submicromolar range.

Characterization of Ganstigmine metabolites in hepatocytes by low- and high-resolution mass spectrometry coupled with liquid chromatography.

In order to deepen the understanding of the metabolism of Ganstigmine, a new acetylcholinesterase inhibitor under evaluation for the treatment of Alzheimer's disease, samples obtained by incubating the drug with female rat hepatocytes were investigated by low-resolution liquid chromatography/tandem mass spectrometry (LC/MS/MS). The results confirmed the formation of most of the phase I metabolites already demonstrated, but also three new species. The combination of high-resolution quadrupole time-of-flight (Q-TOF) LC/MS and LC/MS/MS measurements, and the evaluation of the more reasonable metabolic routes, allowed the identification of the new metabolites as Geneseroline-glucuronide and oxidized and rearranged Ganstigmine. Analogous investigations were made using hepatocytes from male rat and dog, and both gender monkeys and humans, to compare the metabolic patterns. The results did not indicate substantial differences in terms of numbers and abundances of detected metabolites among the considered species, and also between male and female hepatocytes within each species.

Different electrospray tandem mass spectrometric approaches for rapid characterization of phospholipid classes of Curosurf, a natural pulmonary surfactant.

Curosurf is a pulmonary surfactant used in the treatment or prophylaxis of neonatal respiratory distress syndrome. It contains low molecular weight hydrophobic apoproteins and a series of lipids including phosphatidylcholines, lisophosphatidylcholines, phosphatidylethanolamines, sphingomyelins, phosphatidylinositols, phosphatidylglycerols and phosphatidylserines. In the present work, a rapid method to qualitatively map the Curosurf phospholipid classes without prior derivatization or chromatographic separations is described. In particular, a series of specific electrospray tandem mass spectrometric (ES-MS/MS) experiments, i.e. product ion, precursor ion and neutral loss scans, were chosen on the basis of the chemical nature of each phospholipid class and then used to identify single components of the commercial suspension, directly infused into the ion source of the mass spectrometer.