Validation of an ultra high performance liquid chromatography-tandem mass spectrometry method for detection and quantitation of 19 endocrine disruptors in milk

An endocrine disruptor (ED) is an exogenous compound that interferes with the body's endocrine system. Exposure to EDs may result in adverse health effects such as infertility and cancer. EDs are composed of a vast group of chemicals including compounds of natural origin such as phytoestrogens or mycotoxins and a wide range of man-made chemicals such as pesticides. Synthetic compounds may find their way into the food chain where a number of them can biomagnify. Additionally, processing activities and food contact materials may add further to the already existing pool of food contaminants. Thus, our diet is considered to be one of the main exposure routes to EDs. Some precautionary legislation has already been introduced to control production and/or application of some persistent organic pollutants with ED characteristics. However, newly emerging EDs with bioaccumulative properties have recently been reported to appear at lower tiers of the food chain but have not been monitored at the grander scale. Milk and dairy products are a major component of our diet, thus it is important to monitor them for EDs. However, most methods developed to date are devoted to one group of compounds at a time. The UHPLC-MS/MS method described here has been validated according to EC decision 2002/657/EC and allows simultaneous extraction, detection, quantitation and confirmation of 19 EDs in milk. The method calibration range is between 0.50 and 20.0 μg kg with coefficients of determination above 0.99 for all analytes. Precision varied from 4.7% to 23.4% in repeatability and reproducibility studies. Established CCα and CCβ values (0.11-0.67 μg kg) facilitate fast, reliable, quantitative and confirmatory analysis of sub μg kg levels of a range of EDs in milk.

Palladium catalysed tandem cyclisation - Anion capture processes .2. Cyclisation onto alkynes or allenes with hydride capture.

A wide range of palladium catalysed regio- and stereo-specific 5-, 6- and 7-exo-dig mono-, bis- and tris-cyclisation processes of aryl and vinyl halides and allylic acetates are described. The mono- and bis-cyclisation processes terminate in hydride capture from piperidine-formic acid or sodium formate. Addition of TI2CO3 results in alkyne-allene isomerisation and leads, after cyclisation, to 1,3-dienes which give Diels-Alder adducts in good yield. Copyright (C) 1996 Elsevier Science Ltd

PALLADIUM CATALYZED TANDEM CYCLIZATION-ANION CAPTURE PROCESSES .1. BACKGROUND AND HYDRIDE ION CAPTURE BY ALKYL-PALLADIUM AND PI-ALLYL-PALLADIUM SPECIES

A new, wide ranging, synthetically powerful, catalytic tandem cyclisation-anion capture process is proposed which depends on the rate of cyclisation of an organopalladium specifies (RPdX) onto a proximate alkene or diene being significantly faster than anion exchange and reductive elimination in the sequence RPdX --> RPdY --> RY + Pd(0). The catalytic cyclisation - anion capture sequence is illustrated for hydride capture by a wide variety of substrates giving rise to fused- and spiro-, carbo- and hetero-cyclic systems, regio- and stereo-specifically.

PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents

Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft-versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT.

Motorização de fármacos na água bruta e na água para consumo humano por cromatolografia líquida de ultra eficiência associada à espectrometria de massa tandem

Tese de doutoramento, Farmácia (Bromatologia), Universidade de Lisboa, Faculdade de Farmácia, 2014

Characterization of metabolites of sweroside in rat urine using ultra-high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry and NMR spectroscopy

Sweroside, a major active iridoid in Swertia pseudochinensis Hara, is recognized as an effective agent in the treatment of liver injury. Based on previous reports, the relatively short half-life (64 min) and poor bioavailability (approximately 0.31%) in rats suggested that not only sweroside itself but also its metabolites could be responsible for the observed hepato-protective effect. However, few studies have been carried out on the metabolism of sweroside. Therefore, the present study aimed at identifying the metabolites of sweroside in rat urine after a single oral dose (100 mg/kg). With ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC/Q-TOF-MS), the metabolic profile revealed 11 metabolites in rat urine, including phase I, phase II and aglycone-related products. The chemical structures of metabolites were proposed based on accurate mass measurements of protonated or deprotonated molecules and their fragmentation patterns. Our findings showed that the aglycone of sweroside (M05) and its glucuronide conjugate (M06) were principal circulating metabolites in rats. While several other metabolic transformations, occurring via reduction, N-heterocyclization and N-acetylation after deglycosylation, were also observed. Two metabolites (M05 and M06) were isolated from the rat urine for structural elucidation and identifcation of reaction sites. Both M05 and M06 were characterized by 1H, 13C and two-dimensional nuclear magnetic resonance (NMR) spectroscopy. UHPLC/Q-TOF-MS analysis has provided an important analytical platform to gather metabolic profile of sweroside.

Structure-inhibition relationship of phenylethanoid glycosides on angiotensin-converting enzyme using ultra-performance liquid chromatography-tandem quadrupole mass spectrometry

Angiotensin-converting enzyme (ACE) plays a critical role in rennin-angiotensin system. Recently, natural products isolated from herbal medicines revealed inhibitory effects against ACE which suggested their potential activities in regulating blood pressure. In this study, ACE inhibition (ACEI) of 21 phenylethanoid glycosides and related phenolic compounds were investigated by measuring the production of HA a rapid, sensitive, accurate and specific ultra-performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS/MS) method. The test compounds showed different inhibitory potencies on ACE ranging from 5.29 to 95.01% at 50 mM, and the compounds with ACEI higher than 50% were selected for further IC50 determination. The IC50 values were from 0.53 ± 0.04 to 15.035 ± 0.036 mM. The structure-inhibition relationship were then explored and the result showed that cinnamoyl groups played an essential role in ACEI of phenylethanoid glycosides. Furthermore, the sub-structures of increasing ACEI for phenylethanoid glycosides is more hydroxyls and less steric hindrance to chelate the active site Zn2+ of ACE. In summary, our results suggested that phenylethanoid glycosides are a widely available source of anti-hypertensive natural products and the information provided from structure-inhibition relationship study could aid the design of structurally modified phenylethanoid glycosides as anti-hypertensive drugs.

Analysis of six fungicides and one acaricide in still and fortified wines using solid-phase microextraction-gas chromatography/tandem mass spectrometry

A multiresidue gas chromatographic method for the determination of six fungicides (captan, chlorthalonil, folpet, iprodione, procymidone and vinclozolin) and one acaricide (dicofol) in still and fortified wines was developed. Solid-phase microextraction (SPME) was chosen for the extraction of the compounds from the studied matrices and tandem mass spectrometry (MS/MS) detection was used. The extraction consists in a solvent free and automated procedure and the detection is highly sensitive and selective. Good linearity was obtained with correlation coefficients of regression (R2) > 0.99 for all the compounds. Satisfactory results of repeatability and intermediate precision were obtained for most of the analytes (RSD < 20%). Recoveries from spiked wine ranged from 80.1% to 112.0%. Limits of quantification (LOQs) were considerably below the proposedmaximumresidue limits (MRLs) for these compounds in grapes and below the suggested limits for wine (MRLs/10), with the exception of captan.

The development and optimization of a modified single-drop microextraction method for organochlorine pesticides determination by gas chromatography-tandem mass spectrometry

We have developed a new method for single-drop microextraction (SDME) for the preconcentration of organochlorine pesticides (OCP) from complex matrices. It is based on the use of a silicone ring at the tip of the syringe. A 5 μL drop of n-hexane is applied to an aqueous extract containing the OCP and found to be adequate to preconcentrate the OCPs prior to analysis by GC in combination with tandem mass spectrometry. Fourteen OCP were determined using this technique in combination with programmable temperature vaporization. It is shown to have many advantages over traditional split/splitless injection. The effects of kind of organic solvent, exposure time, agitation and organic drop volume were optimized. Relative recoveries range from 59 to 117 %, with repeatabilities of <15 % (coefficient of variation) were achieved. The limits of detection range from 0.002 to 0.150 μg kg−1. The method was applied to the preconcentration of OCPs in fresh strawberry, strawberry jam, and soil.

Mass spectrometry parameters optimization for the 46 multiclass pesticides determination in strawberries with gas chromatography ion-trap tandem mass spectrometry

Multiclass analysis method was optimized in order to analyze pesticides traces by gas chromatography with ion-trap and tandem mass spectrometry (GC-MS/MS). The influence of some analytical parameters on pesticide signal response was explored. Five ion trap mass spectrometry (IT-MS) operating parameters, including isolation time (IT), excitation voltage (EV), excitation time (ET),maximum excitation energy or “q” value (q), and isolationmass window (IMW) were numerically tested in order to maximize the instrument analytical signal response. For this, multiple linear regression was used in data analysis to evaluate the influence of the five parameters on the analytical response in the ion trap mass spectrometer and to predict its response. The assessment of the five parameters based on the regression equations substantially increased the sensitivity of IT-MS/MS in the MS/MS mode. The results obtained show that for most of the pesticides, these parameters have a strong influence on both signal response and detection limit.Using the optimized method, a multiclass pesticide analysis was performed for 46 pesticides in a strawberry matrix. Levels higher than the limit established for strawberries by the European Union were found in some samples.

Analysis of PCBs in soils and sediments by microwave-assisted extraction, headspace-SPME and high resolution gas chromatography with ion-trap tandem mass spectrometry

A procedure for the determination of seven indicator PCBs in soils and sediments using microwave-assisted extraction (MAE) and headspace solid-phase microextraction (HS-SPME) prior to GC-MS/MS is described. Optimization of the HS-SPME was carried out for the most important parameters such as extraction time, sample volume and temperature. The adopted methodology has reduced consumption of organic solvents and analysis runtime. Under the optimized conditions, the method detection limit ranged from 0.6 to 1 ng/g when 5 g of sample was extracted, the precision on real samples ranged from 4 to 21% and the recovery from 69 to 104%. The proposed method, which included the analysis of a certified reference material in its validation procedure, can be extended to several other PCBs and used in the monitoring of soil or sediments for the presence of PCBs.

Enantiomeric fraction evaluation of pharmaceuticals in environmentalmatrices by liquid chromatography-tandem mass spectrometry

The interest for environmental fate assessment of chiral pharmaceuticals is increasing and enantioselective analytical methods are mandatory. This study presents an enantioselective analytical method for the quantification of seven pairs of enantiomers of pharmaceuticals and a pair of a metabolite. The selected chiral pharmaceuticals belong to three different therapeutic classes, namely selective serotonin reuptake inhibitors (venlafaxine, fluoxetine and its metabolite norfluoxetine), beta-blockers (alprenolol, bisoprolol, metoprolol, propranolol) and a beta2-adrenergic agonist (salbutamol). The analytical method was based on solid phase extraction followed by liquid chromatography tandem mass spectrometry with a triple quadrupole analyser. Briefly, Oasis® MCX cartridges were used to preconcentrate 250 mL of water samples and the reconstituted extracts were analysed with a Chirobiotic™ V under reversed mode. The effluent of a laboratory-scale aerobic granular sludge sequencing batch reactor (AGS-SBR) was used to validate the method. Linearity (r2 > 0.99), selectivity and sensitivity were achieved in the range of 20–400 ng L−1 for all enantiomers, except for norfluoxetine enantiomers which range covered 30–400 ng L−1. The method detection limits were between 0.65 and 11.5 ng L−1 and the method quantification limits were between 1.98 and 19.7 ng L−1. The identity of all enantiomers was confirmed using two MS/MS transitions and its ion ratios, according to European Commission Decision 2002/657/EC. This method was successfully applied to evaluate effluents of wastewater treatment plants (WWTP) in Portugal. Venlafaxine and fluoxetine were quantified as non-racemic mixtures (enantiomeric fraction ≠ 0.5). The enantioselective validated method was able to monitor chiral pharmaceuticals in WWTP effluents and has potential to assess the enantioselective biodegradation in bioreactors. Further application in environmental matrices as surface and estuarine waters can be exploited.