Center-of-Mass iso-Energetic Collision-Induced Decomposition in Tandem Triple Quadrupole Mass Spectrometry

Two scan modes of the triple quadrupole tandem mass spectrometer, namely Collision Induced Dissociation Precursor Ion scan and Neutral Loss scan, allow selectively pinpointing, in a complex mixture, compounds that feature specific chemical groups, which yield characteristic fragment ions or are lost as distinctive neutral fragments. This feature of the triple quadrupole tandem mass spectrometer allows the non-target screening of mixtures for classes of components. The effective (center-of-mass) energy to achieve specific fragmentation depends on the inter-quadrupole voltage (laboratory-frame collision energy) and on the masses of the precursor molecular ion and of the collision gas, through a non-linear relationship. Thus, in a class of homologous compounds, precursor ions activated at the same laboratory-frame collision energy face different center-of-mass collision energy, and therefore the same fragmentation channel operates with different degrees of efficiency. This article reports a linear equation to calculate the laboratory-frame collision energy necessary to operate Collision-Induced Dissociation at the same center-of-mass energy on closely related compounds with different molecular mass. A routine triple quadrupole tandem mass spectrometer can operate this novel feature (iso-energetic collision-induced dissociation scan; i-CID) to analyze mixtures of endogenous metabolites by Precursor Ion and Neutral Loss scans. The latter experiment also entails the hitherto unprecedented synchronized scanning of all three quadrupoles of the triple quadrupole tandem mass spectrometer. To exemplify the application of this technique, this article shows two proof-of-principle approaches to the determination of biological mixtures, one by Precursor Ion analysis on alpha amino acid derivatized with a popular chromophore, and the other on modified nucleosides with a Neutral Fragment Loss scan

Establishing health-based biological exposure limits for pesticides : A proof of principle study using mancozeb

Pesticides represent an economical, labor-saving, and efficient tool for pest management, but their intrinsic toxic properties may endanger workers and the general population. Risk assessment is necessary, and biological monitoring represents a potentially valuable tool. Several international agencies propose biological exposure indices (BEI), especially for substances which are commonly absorbed through the skin. Biological monitoring for pesticide exposure and risk assessment seems a natural choice, but biological exposure limits (BEL) for pesticides are lacking. This study aims at establishing equivalent biological exposure limits (EBEL) for pesticides using real-life field data and the Acceptable Operator Exposure Level (AOEL) of mancozeb as the reference. This study included a group of 16 vineyard pesticide applicators from Northern Italy, a subgroup of a more extensive study of 28 applicators. Their exposure was estimated using \u201cpatch\u201d and \u201chand-wash\u201d methodologies, together with biological monitoring of free ethylene-bis-thiourea (ETU) excretion in 24-h pre- and post-exposure urine samples. Modeling was done using univariate linear regression with ETU excretion as the dependent variable and the estimated absorbed dose as the independent variable. The median skin deposition of mancozeb in our study population was 125 \u3bcg, leading to a median absorbed dose of 0.9 \u3bcg/kg. The median post-exposure ETU excretion was 3.7 \u3bcg. The modeled EBEL for mancozeb was 148 \u3bcg of free ETU or 697 \u3bcg of total ETU, accounting for around 75% of the maximum theoretical excretion based on a mass balance model. Although preliminary and based on a small population of low-exposed workers, our results demonstrate a procedure to develop strongly needed biological exposure limits for pesticides

Discovery of unexpected sphingolipids in almonds and pistachios with an innovative use of triple quadrupole tandem mass spectrometry

The densely packed storage of valuable nutrients (carbohydrates, lipids, proteins, micronutrients) in the endosperm of nuts and seeds makes the study of their complex composition a topic of great importance. Ceramides in the total lipid extract of some ground almonds and pistachios were searched with a systematic innovative discovery precursor ion scan in a triple quadrupole tandem mass spectrometry, where iso-energetic collision activated dissociation was performed. Five descriptors were used to search components with different C18 long chain bases containing different structural motifs (d18:0, d18:1, d18:2, t18:0, t18:1). The presence of hexoside unit was screened with a specific neutral loss experiment under iso-energetic collision activated dissociation conditions. The discovery scans highlighted the presence of two specific hexosyl-ceramides with a modified sphingosine component (d18:2) and C16:0 or C16:0 hydroxy-fatty acids. The hexosyl-ceramide with the non-hydroxylated fatty acid seemed specific of pistachios and was undetected in almonds. The fast and comprehensive mass spectrometric method used here can be useful to screen lipid extracts of several more seeds of nutraceutical interest, searching for unusual and/or specific sphingosides with chemically decorated long chain bases

Unambiguous Characterization of p-Cresyl Sulfate, a Protein-Bound Uremic Toxin, as Biomarker of Heart and Kidney Disease

p-Cresyl sulfate is one of the bound uremic toxins whose level increases in the sera of patients with the severity of chronic kidney disease and is therefore used as a standard for clinical investigations. Our first attempts to obtain p-cresyl sulfate led exclusively to the product of sulfonation of the aromatic ring instead of sulfation on the OH moiety. Nevertheless, this initial discouraging result allowed us to handle both p-cresyl sulfate and 2-hydroxy-5-methylbenzenesulfonic acid obtained by different synthetic pathways. Interestingly, the comparison between the two isomers pointed out that the two molecules show the same fragmentation pattern and are indistinguishable by mass spectrometry. They cannot be separated on several commercially available columns. The only difference between the two compounds is a 10-fold higher ionization yield under negative ion electrospray ionization. NMR spectral studies definitely confirmed the different molecular structures. We present here an unambiguous biomimetic synthetic route for p-cresyl sulfate and the spectroscopic characterization of both the compounds by nuclear magnetic resonance and mass spectrometry

Oxidative Stress Markers to Investigate the Effects of Hyperoxia in Anesthesia

Oxygen (O-2) is commonly used in clinical practice to prevent or treat hypoxia, but if used in excess (hyperoxia), it may act as toxic. O-2 toxicity arises from the enhanced formation of Reactive Oxygen Species (ROS) that exceed the antioxidant defenses and generate oxidative stress. In this study, we aimed at assessing whether an elevated fraction of inspired oxygen (FiO(2)) during and after general anesthesia may contribute to the unbalancing of the pro-oxidant/antioxidant equilibrium. We measured five oxidative stress biomarkers in blood samples from patients undergoing elective abdominal surgery, randomly assigned to FiO(2) = 0.40 vs. 0.80: hydroperoxides, antioxidants, nitrates and nitrites (NOx), malondialdehyde (MDA), and glutathionyl hemoglobin (HbSSG). The MDA concentration was significantly higher 24 h after surgery, and the body antioxidant defense lower, in the FiO(2) = 0.80 group with respect to both the FiO(2) = 0.40 group and the baseline values (p <= 0.05, Student's t-test). HbSSG in red blood cells was also higher in the FiO(2) = 0.80 group at the end of the surgery. NOx was higher in the FiO(2) = 0.80 group than the FiO(2) = 0.40 group at t = 2 h after surgery. MDA, the main end product of the peroxidation of polyunsaturated fatty acids directly influenced by FiO(2), may represent the best marker to assess the pro-oxidant/antioxidant equilibrium after surgery

LC-MS/MS-Based Profiling of Tryptophan-Related Metabolites in Healthy Plant Foods

Food plants contain hundreds of bioactive phytochemicals arising from different secondary metabolic pathways. Among these, the metabolic route of the amino acid Tryptophan yields a large number of plant natural products with chemically and pharmacologically diverse properties. We propose the identifier "indolome" to collect all metabolites in the Tryptophan pathway. In addition, Tryptophan-rich plant sources can be used as substrates for the fermentation by yeast strains to produce pharmacologically active metabolites, such as Melatonin. To pursue this technological development, we have developed a UHPLC-MS/MS method to monitor 14 Tryptophan, Tryptamine, amino-benzoic, and pyridine metabolites. In addition, different extraction procedures to improve the recovery of Tryptophan and its derivatives from the vegetal matrix were tested. We investigated soybeans, pumpkin seeds, sesame seeds, and spirulina because of their botanical diversity and documented healthy effects. Four different extractions with different solvents and temperatures were tested, and water extraction at room temperature was chosen as the most suitable procedure to extract the whole Tryptophan metabolites pattern (called by us "indolome") in terms of ease, high efficiency, short time, low cost, and sustainability. In all plant matrices, Tryptophan was the most abundant indole compound, while the pattern of its metabolites was different in the diverse plants extracts. Overall, 5-OH Tryptamine and Kynurenine were the most abundant compounds, despite being 100-1000-fold lower than Tryptophan. Melatonin was undetected in all extracts, but sesame showed the presence of a Melatonin isomer. The results of this study highlight the variability in the occurrence of indole compounds among diverse food plants. The knowledge of Tryptophan metabolism in plants represents a relevant issue for human health and nutrition

Pharmacokinetics and bioavailability of different acetylsalicylic acid formulations assessed by liquid chromatography-tandem mass spectrometry in healthy subjects

Low-dose acetylsalicylic acid (ASA, 100 mg/die) is used in thromboprophylaxis. Enteric-coated formulation (EC-ASA) is commonly used for its lower risk of side effects. Some patients on EC-ASA do not respond appropriately and recent studies showed that poor responsiveness is more frequent with EC-ASA [1]. Aim of this study was to validate a method useful to study the pharmacokinetics (PK) of ASA in healthy subjects treated with two different aspirin formulations

Bioactive phytochemicals of tree nuts. Determination of the melatonin and sphingolipid content in almonds and pistachios

Tree nuts are healthy foods rich in bioactive phytochemicals. Their regular, moderate consumption has been associated with a reduced risk of chronic-degenerative diseases, in the context of a healthy diet and lifestyle. This study aimed to investigate the phytochemical profile of almonds and pistachios, in order to add new elements in the complex scenario of nut chemistry. A LC-MS/MS method was developed to quantify melatonin and ceramides in almonds and pistachios. In general, pistachios were richer in melatonin (2609\u2009\ub1\u20093096 vs 1222\u2009\ub1\u2009500 pg/g) and total ceramides (302\u2009\ub1\u200977 vs 165\u2009\ub1\u200921\u2009pmol/g) than almonds. Among total ceramides fatty acyl homologs, the most represented was the C16:0 species, both for ceramides and dihydroceramides, and both in almonds (37-40%) and pistachios (51-74%). Overall, these results add a piece of information to elucidate the chemical composition of almonds and pistachios and provide a rationale for the nutraceutical potential of nuts in the Mediterranean diet

Pharmacokinetic of myriocin in rabbit’s eyes

Myriocin (Myr) is a suicide inactivator of ceramide synthesis with a complex lipid multifunctional structure. Its biological activity is exerted at very low doses, and thus highly performing quantitative method are needed [1]. The pharmacological development of Myr to modulate ceramide levels also requires currently unavailable ADME information in healthy and pathological animal models

Determination of the serine palmitoyl transferase inhibitor Myriocin by electrospray and Q-trap mass spectrometry

Myriocin, is a potent inhibitor of serine-palmitoyl-transferase, the \ufb01rst and rate-determining enzyme in the sphingolipids biosynthetic pathway. This study developed, validated and applied a LC-MS/MS method to measure Myriocin in minute specimens of animal tissue. The chemical analog 14-OH-Myriocin is used as the internal standard. The two molecules are extracted from the tissue homogenate by solid-phase extraction, separated by gradient reverse-phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection is accomplished by Multiple Reaction Monitoring, employing the most representative transitions: 400@104 and 402@104 for Myriocin and 14-OH-Myriocin, respectively. The typical LoD and LLoQ of the optimized method are 0.9 pmoles/mL (approx. 0.016 pmoles injected) and 2.3 pmoles/mL, respectively, and the method is linear up to 250 pmoles/mL range (r2= 0.9996). The intra-and between-day repeatability affords a CV% 64 7.0. Applications included quantification of Myriocin in mouse lungs after 24 hrs from administration of ~4 nmoles by intra-trachea delivery. Measured levels ranged from 4.11 (median; 2.3-7.4 IQR, n=4) to 11.7 (median; 7.6-22.7 IQR, n=6) pmoles/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (<LoD) to 0.35 pmoles/retina