Nitrogen stable isotope analysis of sulfonamides by derivatization-gas chromatography-isotope ratio mass spectrometry.

The continuous introduction of micropollutants into the environment through livestock farming, agricultural practices, and wastewater treatment is a major concern. Among these pollutants are synthetic sulfonamide antibiotics such as sulfamethoxazole, which are not always fully degraded and pose a risk of fostering antimicrobial resistance. It is challenging to assess the degradation of sulfonamides with conventional concentration measurements. This study introduces compound-specific isotope analysis of nitrogen isotope ratios at natural abundances by derivatization-gas chromatography hyphenated with isotope ratio mass spectrometry (derivatization-GC-IRMS) as a new and more precise method for tracing the origin and degradation of sulfonamides. Here, sulfamethoxazole was used as a model compound to develop and optimize the derivatization conditions using (trimethylsilyl)diazomethane as a derivatization reagent. With the optimized conditions, accurate and reproducible δ15N analysis of sulfamethoxazole by derivatization-GC-IRMS was achieved in two different laboratories with a limit for precise isotope analysis of 3 nmol N on column, corresponding to 0.253 µg non-derivatized SMX. Application of the method to four further sulfonamides, sulfadiazine, sulfadimethoxine, sulfadimidine, and sulfathiazole, shows the versatility of the developed method. Its benefit was demonstrated in a first application, highlighting the possibility of distinguishing sulfamethoxazole from different suppliers and pharmaceutical products.

General Methodologies Toward cis-Fused Quinone Sesquiterpenoids. Enantiospecific Synthesis of the epi-Ilimaquinone Core Featuring Sc-Catalyzed Ring Expansion.

A stereocontrolled approach to the cis-decalin framework of clerodane diterpenes and biologically active quinone sesquiterpenes is reported. Starting from an inexpensive optically pure tetrahydroindanone, Birch reductive alkylation builds two new contiguous chiral centers-one of which is quaternary and all-carbon-substituted. Also featured is a highly regioselective diazoalkane-carbonyl homologation reaction to prepare the 6,6-bicyclic skeleton. Therein, the utility of Sc(OTf)3 as a mild catalyst for formal 1C insertion in complex settings is demonstrated.

Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling.

Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling.

Synthesis of 2-aryl-2H-tetrazoles via a regioselective [3+2] cycloaddition reaction.

A regioselective cycloaddition reaction of arenediazonium salts with trimethylsilyldiazomethane is reported. A series of 2-aryltetrazoles were obtained in good to moderate yields with wide functional group compatibility. Furthermore, this cycloaddition reaction opens the way to build up the versatile intermediate 2-aryl-5-bromotetrazole.

Formal aminocyanation of α,ß-unsaturated cyclic enones for the efficient synthesis of α-amino ketones.

Installation of amino functionality on organic molecules through direct CN bond formation is an important research objective. To achieve this goal, a 1,2-aminocyanation reaction was developed. The reaction occurs through the formation of pyrazolines by means of a formal dipolar cycloaddition of cyclic α,ß-unsaturated ketones with lithium trimethylsilyldiazomethane followed by novel protonolytic N-N bond cleavage under mild conditions. This two-step process provides a diverse array of structurally complex free and mono-alkylated α-amino ketones in excellent yields.

An integrated approach to evaluate acetamiprid-induced oxidative damage to tRNA in human cells based on oxidized nucleotide and tRNA profiling.

Acetamiprid is poisonous to mammals due to severe acetamiprid-induced oxidative stress that could cause mitochondrial dysfunctions, lipid and protein oxidation, inflammation, apoptosis, and DNA damage. Evidence has accumulated for the role of oxidative stress in changing structures and functions of transfer RNAs (tRNAs) by inducing tRNA cleavage, reprogramming tRNA modifications and impairing aminoacyl-tRNA synthetase editing sites. However, the impact of acetamiprid-induced oxidative stress on tRNA is still unknown. Here, we investigated the effects of acetamiprid on cell viability, reactive oxygen species (ROS) levels, DNA damage, cellular oxidized nucleotide concentrations, and oxidative damage to tRNA in HepG2 cells and LO2 cells. Acetamiprid can cause the significant increment of ROS and DNA oxidative damage. In this study, an integrated approach was established to simultaneously study the network of oxidized nucleotides and explore the tRNA oxidative damage after acetamiprid exposure. A simple and high-throughput liquid chromatography with tandem mass spectrometry (LC-MS/MS) method coupled with (trimethylsilyl)diazomethane (TMSD) derivatization was successfully developed to quantify 12 cellular oxidized nucleotides that cannot be detected using traditional detection methods because of the huge interferences from naturally abundant nucleotides. Meanwhile, the accumulation rate and the locating sites of 8-oxo-2, 7-dihydro-guanine (8-oxo-G) in tRNA were inspected using the established N-(tert-Butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) labeling-based tRNA profiling method. After acetamiprid treatment, the increment of oxidized nucleoside triphosphates is smaller than that of their corresponding mono- and diphosphates, as well as the dephosphorylated nucleosides, on account of the existence of sanitization enzymes. Several tRNA fragments, CUC[m1A]Gp, CACGp, [Cm]C[m2G]p, and DDGp, are significantly downregulated in acetamiprid-treated HepG2 cells, while only [Cm]C[m2G]p in acetamiprid-treated LO2 cells. According to the profiling results, the significantly changed fragment CUC[m1A]Gp might be caused by the oxidation of guanine (G) to form 8-oxo-G at position 15 in human tRNAphe([Gm]AA), providing more information about the effect of oxidized nucleobases on tRNA's functions.

A method for determining valproic acid in human whole blood and urine via gas chromatography-mass spectrometry and small-scale inter-laboratory trial.

A simple and cost-effective method for analyzing valproic acid (VPA) in biological samples was developed. VPA was extracted in methyl tertiary-butyl ether (MTBE) and derivatized using trimethylsilyldiazomethane. The MTBE extract was analyzed by gas chromatography-mass spectrometry (GC-MS). The extraction recovery in human whole blood and urine was over 90 %, with good linearity in the range of 1.0 to 250 µg/mL of VPA. The RSD for 2.0, 20, and 200 µg/mL VPA in whole blood ranged from 0.9 to 4.7 % for intra-day and 1.5 to 5.9 % for inter-day. The RSD for 2.0, 20, and 200 µg/mL VPA in urine ranged from 1.9 to 2.6 % for intra-day and 1.2 to 2.9 % for inter-day. As a preliminary cross-validation study, a cross-check was conducted using blinded concentration samples. The results demonstrated that the assay data of the two laboratories were comparable.

Analysis of trace phosphonates in authentic water samples by pre-methylation and LC-Orbitrap MS/MS.

Phosphonate is an important phosphorous species in the effluent of wastewater treatment plant (WWTP), contributing to eutrophication and interfering with phosphate removal in WWTP. It is particularly difficult to determine phosphonates in samples of complex solution chemistry, resulting in very limited information on their presence in environmental matrices. Herein, we proposed a sensitive method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine six quantitatively most important phosphonates even at the ng/L level, i.e., 2-phosphonobutane-1,2,4-tricarboxylic acid, 1-hydroxyethane 1,1-diphosphonic acid, nitrilotris(methylene phosphonic acid), ethylenediamine tetra(methylene phosphonic acid), hexamethylenediamine tetra(methylene phosphonic acid) and diethylenetriamine penta(methylene phosphonic acid). Trimethylsilyldiazomethane (TMSCHN2) derivatization of the target phosphonates is pre-requisite since it could greatly increase the sensitivity up to 2-3 orders of magnitude over direct analysis of the virgin ones. The sample pretreatment methods (including ion exchange and solid phase extraction(SPE)), the derivatization procedures, and the LC-MS/MS conditions were systematically optimized. The limits of quantitation for the six phosphonates in the background of tap water ranged from 1.4 µg/L to 57 µg/L for direct analysis, and from 5.0 ng/L to 200 ng/L for SPE enabled pre-concentration analysis, respectively. The reliability of the proposed method was successfully validated by analysis of authentic water samples collected from one river and three WWTPs (0.088-7200 µg/L phosphonates) with satisfactory recoveries (72-126%). To the best of knowledge, this is the first report on quantification of phosphonates in environmental samples in China.

Validation of a novel and rapid method for the simultaneous determination of some phenolic organohalogens in human serum by GC-MS.

Over the last decades, more and more studies focused on the impact of endocrine disruptors on the environment and human health. Among them, phenolic organohalogens (POHs) are a particular concern because of their structural resemblance with natural hormones. There are different methods that are known to quantify these compounds in human serum, however, the current extraction techniques are long, fastidious and using harmfull chemicals such as diazomethane and sulfuric acid. Consequently, we developed an alternative, sensitive and faster method to simultaneously quantify pentachlorophenol (PCP), tetrabromobisphenol A (TBBPA), 4 bromophenols, 7 hydroxypolychlorinated biphenyls (OH-PCBs) and 3 hydroxy-polybrominated diphenyl ether (OH-PBDEs) in human serum sample. The clean-up and the enrichment of the sample were performed in a single extraction step using strong anion-exchange solid phase cartridge. After a rapid liquid-liquid extraction step to remove acidic traces, the extract was derivatized using trimethylsilyldiazomethane (TMSD) and finally analyzed by a gas-chromatograph coupled with an electron negative capture chemical ionization source combined with a triple quadrupole mass spectrometer (GC-ENCI-MS) operating in single ion monitoring. The whole procedure was validated according to the total error approach. The inter and intra assay precision were demonstrated to be lower than 20% and the relative bias to be lower than 15% in the dosing range of concentrations. The limit of quantification (LOQ) ranged from 2pgmL-1 and 5pgmL-1, except for the PCP (44.6pgmL-1) and for the 2,4,6-tribromophenol (49.6pgmL-1). Finally, the method was successfully applied to measure the POH background contamination in serum samples collected from 20 Belgian blood donors recruited in CHU Mont-Godinne (Namur, Belgium) aged between 21 and 69 years old.

On-cartridge derivatization coupled with solid-phase extraction for the ultra-sensitive determination of minodronic acid in human plasma by LC-MS/MS method.

Minodronic acid (MA) is a third-generation bisphosphonate (BP). Its high potency allows lower doses to be administered in clinical settings compared with other BPs, which results in extremely low systemic exposure. Therefore, it is essential to develop an ultra-sensitive bioassay for pharmacokinetics studies of MA. In this work, we used on-cartridge derivatization of MA with trimethylsilyldiazomethane to extract MA from plasma samples and improve its LC-MS/MS behavior. The reaction produced a known derivative, tetramethylated MA, and a novel derivative, pentamethylated MA (PMMA). PMMA exhibited a better signal-to-noise ratio, and was monitored for the quantification of MA. However, the derivatization yield of d4-PMMA was much lower and more variable than that of PMMA, which decreased the effectiveness of its correction function as an internal standard. Therefore, a two-cycle derivatization approach was introduced to increase its yield and improve the reproducibility. The calibration curves of MA showed good linearity over the range of 10.0-1000 pg/mL. A lower limit of quantification of 10.0 pg/mL was achieved with acceptable precision (<10.5%) and accuracy (5.0%). The intra- and inter-batch precision of quality control samples was <9.5%, and the accuracy ranged from -2.8% to 0.6%. The stability results showed that MA was stable in human plasma for 6h at room temperature (25°C), for 115 days at -20°C, during three freeze/thaw cycles (from -20°C to 25°C), and in post-preparative samples for 24h at 4°C. The method was successfully used to characterize the pharmacokinetic profile of MA following an oral dose of 1.0mg MA hydrate to healthy volunteers (n=12). The proposed derivatization procedure was also extended to measure other BPs (risedronic acid and zoledronic acid) in human plasma at low pg/mL.

Evaluation of four derivatization methods for the analysis of fatty acids from green leafy vegetables by gas chromatography.

Green leafy vegetables are valuable secondary sources of nutrients, including lipids, commonly consumed in developing countries. However, method development for the analysis of fatty acids is usually focused on the animal lipid samples, rarely including natural plant extracts. Hence, the usefulness of four derivatization methods for the gas chromatographic analysis of plant lipids was studied. Methylation using 10% solution of BF3 in methanol and 2.0M solution of (trimethylsilyl)diazomethane (TMSD) in hexane, trimethylsilylation and tert-butyldimethylsilylation were compared using lipid standards and extracts from the leaves of Solanum macrocarpon and S. melongena after saponification. While silylation was found effective and precise using lipid standards, it initially did not perform well in the analysis of plant lipids due to the presence of transesterification products in samples. Optimization of the hydrolysis conditions resulted in an effective analysis of these derivatives, but poor separation of FA(18:0) from unsaturated FA(18:X) compounds and the presence of larger amounts of interferences disqualified the use silylation for the analysis of plant fatty acids in applied analytical conditions. Methylation using TMSD gave more precise quantitative results when compared to BF3/MeOH method. Also, it produced a significantly lower amount of interferences when applied to plant lipid samples. Additionally, the TMSD-based method is simple, safe and less time-consuming when compared to other procedures. Thus, we suggest using TMSD-based methylation as a method of choice in the GC analysis of plant-derived fatty acids.