Acetylcholinesterases from Leaf-Cutting ant Atta sexdens: Purification, Characterization, and Capillary Reactors for On-Flow Assays.

Acetylcholinesterase (AChE) is responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine (ACh) leading to acetate and choline (Ch) release. The inhibition of AChE produces a generalized synaptic collapse that can lead to insect death. Herein we report for the first time the isolation of two AChEs from Atta sexdens which were purified by sulphate ammonium precipitation followed by ion exchange chromatography. AsAChE-A and AsAChE-B enzymes have optimum pH of 9.5 and 9.0 and higher activities in 30/50°C and 20°C, respectively, using acetylthiocholine (ATCh) as substrate. Immobilized capillary enzyme reactors (ICERs) were obtained for both enzymes (AsAChE-A-ICER and AsAChE-B-ICER) and their activities were measured by LC-MS/MS through hydrolysis product quantification of the natural substrate ACh. The comparison of activities by LC-MS/MS of both AChEs using ACh as substrate showed that AsAChE-B (free or immobilized) had the highest affinity. The inverse result was observed when the colorimetric assay (Elman method) was used for ATCh as substrate. Moreover, by mass spectrometry and phylogenetic studies, AsAChE-A and AsAChE-B were classified as belonging to AChE-2 and AChE-1 classes, respectively.

Prostaglandins E2 and F2α levels in human menstrual fluid by online Solid Phase Extraction coupled to Liquid Chromatography tandem Mass Spectrometry (SPE-LC-MS/MS).

This paper reports an online SPE-LC-MS/MS method for the simultaneous quantification of prostaglandins (PGE2 and PGF2α) in menstrual fluid samples. To meet this goal human peripheral serum was used as surrogate matrix. The analytes were trapped on an OASIS HLB cartridge for 3 min, for sample cleanup and enrichment, and then transferred during only 42 s to an HSS T3 C18 analytical column, for separation and analysis. Prostaglandins (PGs) were detected by selected reaction monitoring in negative ion mode, PGE2 (m/z 351 → 315) and PGF2α (m/z 353 → 193) using isotope-labeled internal standard (PGE2-d4, m/z 355 → 319). The concentration linear range was of 10.34-1.034 ng mL-1 and the lower limit of quantification (LLOQ) was 10.34 ng mL-1 for both PGs. Validation parameters were successfully assessed according to the European Medicines Agency guideline (EMA), also comprising the FDA normative. The method showed no matrix effect and process efficiency around 100%, in addition to only 15 min of analysis time with lower solvent consumption. The method application was carried out using two menstrual fluid sample groups: control (n = 15) and treatment group (n = 7; samples from women that used Tahiti lemon juice). The PGF2α levels were found to be higher in treated group than in control group (p ≤ 0.05), denoting an effect of the intake of Tahiti lemon juice on the menstrual inflammatory process. The on-line method herein reported could be useful for the analysis of PGs from large research studies.

Leveraging the cruzain S3 subsite to increase affinity for reversible covalent inhibitors.

Cruzain is the major cysteine protease of Trypanosoma cruzi, the etiological agent of Chagas disease. Reversible covalent cruzain inhibitors can block the steps of cell differentiation in the parasite and kill the organism. To this end, the description of how inhibitors modified at the P2/P3 positions lead to analogs with greater cruzain affinity to the S2/S3 subsites is of fundamental importance. Albeit many efforts are being employed in the characterization of the interaction processes with S2 subsite, little is known about the cruzain S3 subsite. In this work, we show a brief but consistent study to identify favorable substitutions in P3 of dipeptidyl nitriles that increase cruzain affinity. Using molecular dynamics simulations, we have identified some dipeptidyl nitrile analogs with modifications at P3 position that had higher cruzain inhibition than the original unsubstituted compound. A matched molecular pair analysis shows the importance of including a chlorine atom in the P3-meta position. The modifications implemented in P3 are confirmed when profiling the thermodynamic parameters via isothermal titration calorimetry. The classical enthalpy-entropy compensation phenomenon, in which enthalpy changes are counterbalanced by entropy results in a small modification of ΔG. The inclusion of the chlorine atom in the P3-meta position results in the highest reduction of the detrimental entropic contribution observed in P3.

Simultaneous quantification of fluoxetine and norfluoxetine in colostrum and mature human milk using a 2-dimensional liquid chromatography-tandem mass spectrometry system.

A two-dimensional liquid chromatography system coupled to triple quadrupole tandem mass spectrometer (2D LC-MS/MS) was employed for the determination of fluoxetine (FLU) and norfluoxetine (N-FLU) in colostrum and mature milk by direct sample injection. With a run time of 12 min representing a gain in throughput analysis, the validated methods furnished selectivity, extraction efficiency, accuracy, and precision in accordance with the criteria preconized by the European Medicines Agency guidelines. With a linear range of 3.00-150 ng/mL for FLU and 4.00-200 ng/mL for N-FLU they were applied to the analysis of colostrum and mature milk samples from nursing mothers. The paper discusses the differences and similarity of sample preparation for this two sample matrices. The herein reported methods are an advance in sample preparation procedures providing waste reduction and a sustainable approach.

Bioanalytical challenge: A review of environmental and pharmaceuticals contaminants in human milk.

An overview of bioanalytical methods for the determination of environmental and pharmaceutical contaminants in human milk is presented. The exposure of children to these contaminants through lactation has been widely investigated. The human milk contains diverse proteins, lipids, and carbohydrates and the concentration of these components is drastically altered during the lactation period providing a high degree of an analytical challenge. Sample collection and pretreatment are still considered the Achilles' heel. This review presents liquid chromatographic methods developed in the last 10 years for this complex matrix with focuses in the extraction and quantification steps. Green sample preparation protocols have been emphasized.

Simultaneous enantioselective quantification of fluoxetine and norfluoxetine in human milk by direct sample injection using 2-dimensional liquid chromatography-tandem mass spectrometry.

A two-dimensional liquid chromatography system coupled to triple quadrupole tandem mass spectrometer (2D LC-MS/MS) was employed for the simultaneously quantification of fluoxetine (FLX) and norfluoxetine (NFLX) enantiomers in human milk by direct injection of samples. A restricted access media of bovine serum albumin octadecyl column (RAM-BSAC18) was used in the first dimension for the milk proteins depletion, while an antibiotic-based chiral column was used in the second dimension. The results herein described show good selectivity, extraction efficiency, accuracy, and precision with limits of quantification in the order of 7.5ngmL(-1)for the FLX enantiomers and 10.0ngmL(-1) for NFLX enantiomers. Furthermore, it represents a practical tool in terms of sustainability for the sample preparation of such a difficult matrix.

Quantification of ampicillin in bovine milk by coupled-column ultrahigh-performance liquid chromatography-tandem mass spectrometry.

This work reports the use of two-dimensional (2D) liquid chromatography system coupled with a tandem mass spectrometry for the quantification of ampicillin in bovine milk. A restrict access media column (RAM-BSA C(8) , 50 × 2.1 mm, Luna, 10 µm, 100 Å) was used in the first dimension in order to exclude macromolecules, while an ACQUITY UPLC BEH C(18) (50 × 2.1 mm, 1.7 µm) column was used in the second dimension. Three different channels of selected reaction monitoring (SRM) were used: 350 > 106 m/z, 350 > 160 m/z, and 350 > 192 m/z. The first transition was used for the quantification (higher intensity), and latter two for confirmation. The developed method is simple and requires a total analysis time of only 14 min/sample. The sample treatment involved only a centrifugation step for 20 min. The validated method has been successfully applied to monitor AMP residues in raw milk samples. To our knowledge, this is the first study to report the use of ultrahigh-performance liquid chromatography (UHPLC) in 2D configuration.