A 30 year old man was found with no signs of life in front of the house. The cyanide concentration in blood and urine was determined five years after the man\'s death. What is more, a stability study was conducted for 730 days in an authentic casework blood sample. Sample preparation procedure included precipitation with methanol:water mixture, solid phase extraction (SPE) and derivatization with the use of PFB-Br (pentafluorobenzyl bromide). The sample was analyzed using GC-QqQ-MS/MS (gas chromatopraphy coupled with tandem mass spectrometry) isotope dilution method. Separation was done using a SH-RXI-5MS column (30 m x 0.25 mm, 0.25 µm). Detection of PFB-CN and PFB-13CN was achieved using a triple-quadrupole mass spectrometer with an electron ionization (EI) ion source in multiple reaction monitoring (MRM) mode. After 5 years from the man\'s death, cyanide concentration was: 1900 ng/mL in blood and 500 ng/mL in urine. Stability study performed in an authentic blood sample 6 and 7 years after the man\'s death revealed cyanide concentrations of 1898.2 ng/mL and 1618.7 ng/mL, respectively. While spectrophotometric and colorimetric methods recorded both decrease and increase in cyanide concentration over time, newer chromatographic methods mainly indicate a decrease. The studies presented in this paper seem to confirm this trend. However, in order to interpretate the results of cyanide concentration in biological material reliably, more research is still necessary.

In recent years, agonists of the 5-HT2A receptor have gained increasing attention for their potential therapeutic use to treat psychological disorders such as anxiety and depression. Here, we report the development and validation of an LC-MSMS based analytical method for the quantification of the novel selective 5-HT2A agonist 25CN-NBOH in rat plasma and brain. As simple and efficient sample clean-up we applied the Phree Phospholipid Removal approach from Phenomenex, which is particularly novel for brain samples. In order to investigate the metabolic stability of 25CN-NBOH in vitro biotransformation studies with recombinant enzymes and human liver microsomes were conducted. Several biotransformation products and pathways could be identified. Based on the in vitro study one of the putative metabolites (2C-CN) was included in the analytical method development. To test the methods applicability 25CN-NBOH was quantified in plasma and brain samples from a pharmacokinetic in vivo study with Wildtype Long Evans rats. Both the in vitro metabolism data as well as the in vivo PK data suggest that 25CN-NBOH is susceptible to metabolism, but is degraded slower and is more stable compared to other NBOMe\'s investigated to date. The developed analytical method might serve as basis to include further 25CN-NBOH metabolites. It is expected to facilitate further preclinical and clinical investigations of 25CN-NBOH in biological matrices.

An analytical method for the simultaneous quantitation of ten trichothecenes of type A (HT-2 toxin, T-2 toxin, diacetoxyscirpenol, and neosolaniol) and type B (3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, deoxynivalenol, deoxynivalenol-3-glucoside, nivalenol, and fusarenon-X) in feed has been developed using liquid chromatography with tandem mass spectrometry. Mycotoxins extracted twice from samples using aqueous acetonitrile were purified using a multifunctional clean-up column, followed by a phospholipid removal column. Trichothecenes were analysed using liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry. The extraction efficiency of the mycotoxins and the repeatability of some were improved by repeated extractions. Ionization enhancement (signal enhancement) of some mycotoxins was improved by using the phospholipid removal column at the clean-up step. Spike and recovery tests of trichothecenes were conducted on maize, barley, soybean meal, rapeseed meal, and formula feeds (for starting broiler chicks, suckling pigs, and beef cattle). The mean recovery values were 70.6-119% with relative standard deviations < 17%. The limit of quantification and the limit of detection of our method were 20 and 6 μg/kg, respectively, for 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol; 10 and 3 μg/kg, respectively, for T-2 toxin, deoxynivalenol, and fusarenon-X; and 5 and 2 μg/kg, respectively, for nivalenol and the remaining mycotoxins.

In this study, a reliable ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method coupled with an easy, fast and effective sample pretreatment procedure was developed for simultaneous determination of amitraz, chlordimeform, formetanate and their metabolites in human blood. With the procedures of protein precipitation and a phospholipid-removal step, the endogenous compound interference was significantly reduced, and matrix effects were significantly reduced. The linear ranges of matrix-matched standard curves were from 0.5 to 1000 ng/mL with coefficients of determination >0.996. Very low limits of detection (0.05-0.12 ng/mL) and limits of quantitation (0.15-0.4 ng/mL) were achieved. Reasonable recoveries ranging from 88.1 to 103.5% were obtained. The intra-day RSDs ranging from 3.2 to 8.6% and inter-day RSDs ranging from 4.8 to 9.2% indicated good precision. With the introduction of a phospholipid-removal step, the ME ranged from 90.1 to 98.5%. The established method was successfully applied to the analysis of a blood sample from a formetanate poisoning case. This method possesses the advantages of high sensitivity, reduced matrix effects and rapidity.

OBJECTIVE: To develop a method for determination of bisphenol A in human blood by fast phospholipid removal solid phase extraction method-ultra performance liquid chromatography tandem mass spectrometry( UPLC-MS/MS).
METHODS: Enzymatic hydrolysis( β-glucuronidase/arylsulfatase) and phospholipid removal solid phase extraction were used to treat the blood samples under the acidic condition, a Infinity Lab Poroshell 120 PFP column( 100 mm × 3. 0 mm, 2. 7 μm) was used for LC separation, ESI negative ion scan was used with multiple reaction monitoring( MRM) mode.
RESULTS: The calibration curve was linear in the range of 0. 1-100 ng/mL for bisphenol A with correlation coefficients more than 0. 999. The limit of detection was 0. 05 ng/mL, the limit of quantity was 0. 15 ng/mL. The recoveries of the method for bisphenol A at three spiked levels of 0. 5, 5 and 50 ng/mL ranged from 87. 3%to 112. 1%. The relative standard deviation( RSD) of intra and inter day were range from3. 3%-8. 2%, 4. 9%-10. 7%( n = 6), respectively.
CONCLUSIONS: The method is successfully applied in the analysis of bisphenol A in human blood with its simple operation, sensitivity and accuracy.

Tranexamic acid is a widely used antifibrinolytic drug but its pharmacology and pharmacokinetics remains poorly understood. Owing to the recent knowledge on phospholipid-induced matrix effects during human plasma analysis, our aim was to develop a liquid chromatography-mass spectrometry method for the quantitation of tranexamic acid after efficient sample clean-up. Sample preparation consisted in phospholipid removal and protein precipitation. Hydrophilic interaction liquid chromatography was used and the detection was achieved with multiple reaction monitoring. The method was validated according to the European Medicine Agency guideline in the range 1.0-1000.0μg/mL. The performance of the method was excellent with a precision in the range 1.2-3.0%, an accuracy between 88.4 and 96.6% and a coefficient of variation of the internal standard-normalized matrix factor below 6.7%. This method is suitable for the quantification of tranexamic acid in the wide range of concentrations observed during clinical studies, with all the advantages related to phospholipid removal.

Enzymatic degumming using phospholipase C (PLC) enzymes may be used in environmentally friendly processes with improved oil recovery yields. In this work, phosphatidylinositol-specific phospholipase C (PIPLC) candidates obtained from an in silico analysis were evaluated for oil degumming. A PIPLC from Lysinibacillus sphaericus was shown to efficiently remove phosphatidylinositol from crude oil, and when combined with a second phosphatidylcholine and phosphatidylethanolamine-specific phospholipase C, the three major phospholipids were completely hydrolyzed, providing an extra yield of oil greater than 2.1%, compared to standard methods. A remarkably efficient fed-batch Escherichia coli fermentation process producing ∼14 g/L of the recombinant PIPLC enzyme was developed, which may facilitate the adoption of this cost-effective oil-refining process.

Generic Parallel Artificial Liquid Membrane Extraction (PALME) methods for non-polar basic and non-polar acidic drugs from human plasma were investigated with respect to phospholipid removal. In both cases, extractions in 96-well format were performed from plasma (125μL), through 4μL organic solvent used as supported liquid membranes (SLMs), and into 50μL aqueous acceptor solutions. The acceptor solutions were subsequently analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using in-source fragmentation and monitoring the m/z 184→184 transition for investigation of phosphatidylcholines (PC), sphingomyelins (SM), and lysophosphatidylcholines (Lyso-PC). In both generic methods, no phospholipids were detected in the acceptor solutions. Thus, PALME appeared to be highly efficient for phospholipid removal. To further support this, qualitative (post-column infusion) and quantitative matrix effects were investigated with fluoxetine, fluvoxamine, and quetiapine as model analytes. No signs of matrix effects were observed. Finally, PALME was evaluated for the aforementioned drug substances, and data were in accordance with European Medicines Agency (EMA) guidelines.

An on-line TFC (Turbulent Flow Chromatography) clean up procedures coupled with UHPLC-MS/MS (Ultra High Performance Liquid Chromatography Mass Spectrometry) multi-residue method was developed for the simultaneous determination of 8 perfluroalkyl carboxylic acids (PFCA, from 5 to 12 carbon atoms) and 3 perfluoroalkyl sulfonic acids (PFSA, from 4 to 8 carbon atoms) in environmental solid matrices. Fast sample preparation procedure was based on a sonication-assisted extraction with acetonitrile. Phospholipids in biological samples were fully removed by an off-line SPE purification before injection, using HybridSPE(®) Phospholipid Ultra cartridges. The development of the on-line TFC clean-up procedure regarded the choice of the stationary phase, the optimization of the mobile phase composition, flow rate and injected volume. The validation of the optimized method included the evaluation of matrix effects, accuracy and reproducibility. Signal suppression in the analysis of fortified extracts ranged from 1 to 60%, and this problem was overcome by using isotopic dilution. Since no certified reference materials were available for PFAS in these matrices, accuracy was evaluated by recoveries on spiked clam samples which were 98-133% for PFCAs and 40-60% for PFSAs. MLDs and MLQs ranged from 0.03 to 0.3ngg(-1) wet weight and from 0.1 to 0.9ngg(-1) wet weight respectively. Repeatability (intra-day precision) and reproducibility (inter-day precision) showed RSD from 3 to 13% and from 4 to 27% respectively. Validated on-line TFC/UHPLC-MS/MS method has been applied for the determination of perfluoroalkyl acids in different solid matrices (sediment, fish, bivalves and bird yolk).

N-acetylneuraminic acid (Neu5Ac or NANA) is the most predominant sialic acid in mammals. As a terminal component in many glycoproteins and glycolipids, sialic acid is believed to be an important biomarker related to various diseases. Its precursor, N-acetylmannosamine (ManNAc), is being investigated as a potential treatment for GNE myopathy. In this work, we developed two highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the quantitation of ManNAc and free Neu5Ac in human plasma. A fit-for-purpose approach was adopted during method validation and sample analysis. To measure the endogenous compounds and overcome the interference from plasma samples, a surrogate matrix that contained 5% bovine serum albumin (BSA) was used for the preparation of calibration standards and certain levels of quality control (QC) samples. QC samples at higher concentrations were prepared in the authentic matrix (human plasma) to best mimic incurred samples. For both methods, an Ostro 96-well phospholipid removal plate was used for sample extraction, which efficiently removed the phospholipids from the plasma samples prior to LC injection, eliminated matrix effect, and improved sensitivity. Chromatographic separation was achieved using hydrophilic interaction chromatography (HILIC) and gradient elution in order to retain the two polar compounds. The lower limit of quantitation (LLOQ) for ManNAc and Neu5Ac was 10.0 and 25.0ng/mL, respectively. The overall accuracy of the two assays was within 100%±8.3% based on three levels of QC samples. Inter- and intra-run precision (coefficient of variation (%CV)) across three analytical runs was less than 6.7% for ManNAc and less than 10.8% for Neu5Ac. These methods have been validated to support clinical studies.