Endocannabinoid signaling in the central nervous system.

It is hard to overestimate the influence of the endocannabinoid signaling (ECS) system on central nervous system (CNS) function. In the 40 years since cannabinoids were found to trigger specific cell signaling cascades, studies of the ECS system continue to cause amazement, surprise, and confusion! CB1 cannabinoid receptors are expressed widely in the CNS and regulate cell-cell communication via effects on the release of both neurotransmitters and gliotransmitters. CB2 cannabinoid receptors are difficult to detect in the CNS but seem to "punch above their weight" as compounds targeting these receptors have significant effects on inflammatory state and behavior. Positive and negative allosteric modulators for both receptors have been identified and examined in preclinical studies. Concentrations of the endocannabinoid ligands, N-arachidonoylethanolamine and 2-arachidonoylglycerol (2-AG), are regulated by a combination of enzymatic synthesis and degradation and inhibitors of these processes are available and making their way into clinical trials. Importantly, ECS regulates many essential brain functions, including regulation of reward, anxiety, inflammation, motor control, and cellular development. While the field is on the cusp of preclinical discoveries providing impactful clinical and therapeutic insights into many CNS disorders, there is still much to be learned about this remarkable and versatile modulatory system.

Coupling Stable Isotope Labeling and Liquid Chromatography-Trapped Ion Mobility Spectrometry-Time-of-Flight-Tandem Mass Spectrometry for De Novo Mosquito Ovarian Lipid Studies.

There is a need to better understand lipid metabolism during mosquito ovarian development. Lipids are the major source of energy supporting ovarian follicles development in mosquitoes. In this paper, we describe the complementary use of stable isotope labeling (SIL) and high-resolution mass spectrometry-based tools for the investigation of de novo triglycerides (TG) and diglycerides (DG) during the ovarian previtellogenic (PVG) stage (4-6 days posteclosion) of female adult Aedes aegypti. Liquid chromatography coupled to high-resolution trapped ion mobility spectrometry-parallel accumulation sequential fragmentation-time-of-flight tandem mass spectrometry (LC-TIMS-PASEF-TOF MS/MS) allowed the separation and quantification of nonlabeled and 2H/13C-labeled TG and DG species. Three SIL strategies were evaluated (H2O/2H2O with 50:50 and 95:5 mixtures, 13C-sucrose, and 13C-glucose). Results showed wide applicability with no signs of lipid ovarian impairment by SIL induced toxicity. The analytical workflow based on LC-TIMS-TOF MS/MS provided high confidence and high reproducibility for lipid DG and TG identification and SIL incorporation based on their separation by retention time (RT), collision cross section (CCS), and accurate m/z. In addition, the SIL fatty acid chain incorporation was evaluated using PASEF MS/MS. The 2H/13C incorporation into the mosquito diet provided information on how TG lipids are consumed, stored, and recycled during the PVG stage of ovarian development.

Sexual dimorphism of diapause regulation in the hemipteran bug Pyrrhocoris apterus.

Diapause is one of the major strategies for insects to prepare for and survive harsh seasons. In females, the absence of juvenile hormone (JH) is a hallmark of adult reproductive diapause, a developmental arrest, which is much less characterized in males. Here we show that juvenile hormone III skipped bisepoxide (JHSB3) titers in hemolymph remarkably differ between reproductive males and females of the linden bug Pyrrhocoris apterus, whereas no JH was detected in diapausing adults of both sexes. Like in females, ectopic application of JH mimic effectively terminated male diapause through the canonical JH receptor components, Methoprene-tolerant and Taiman. In contrast to females, long photoperiod induced reproduction even in males with silenced JH reception or in males with removed corpus allatum (CA), the JH-producing gland. JHSB3 was detected in the accessory glands (MAG) of reproductive males, unexpectedly, even in males without CA. If there is a source of JHSB3 outside CA or a long-term storage of JHSB3 in MAGs remains to be elucidated. These sex-related idiosyncrasies are further manifested in different dynamics of diapause termination in P. apterus by low temperature. We would like to propose that this sexual dimorphism of diapause regulation might be explained by the different reproductive costs for each sex.

Epoxidation of juvenile hormone was a key innovation improving insect reproductive fitness.

Methyl farnesoate (MF) plays hormonal regulatory roles in crustaceans. An epoxidated form of MF, known as juvenile hormone (JH), controls metamorphosis and stimulates reproduction in insects. To address the evolutionary significance of MF epoxidation, we generated mosquitoes completely lacking either of the two enzymes that catalyze the last steps of MF/JH biosynthesis and epoxidation, respectively: the JH acid methyltransferase (JHAMT) and the P450 epoxidase CYP15 (EPOX). jhamt-/- larvae lacking both MF and JH died at the onset of metamorphosis. Strikingly, epox-/- mutants, which synthesized MF but no JH, completed the entire life cycle. While epox-/- adults were fertile, the reproductive performance of both sexes was dramatically reduced. Our results suggest that although MF can substitute for the absence of JH in mosquitoes, it is with a significant fitness cost. We propose that MF can fulfill most roles of JH, but its epoxidation to JH was a key innovation providing insects with a reproductive advantage.

Comprehensive Screening of Polycyclic Aromatic Hydrocarbons and Similar Compounds Using GC-APLI-TIMS-TOFMS/GC-EI-MS.

In the present work, a novel workflow based on complementary gas-phase separations for the identification of isomeric PAHs from complex mixtures is described. This is the first report on the coupling of gas chromatography (GC), atmospheric pressure laser ionization (APLI), and trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) for the characterization of polycyclic aromatic hydrocarbons. Over a hundred known unknowns are uniquely identified based on the molecular ion retention indices I (5%), mobility (RSD < 0.6% and R = 50-90 with Sr = 0.18 V/ms), mobility-based theoretical candidate assignment (<3%), accurate mass chemical formula assignment (<2 ppm), and electron impact fragmentation pattern and database search. The advantages of theoretical modeling of PAHs and similar compounds were evaluated using candidate structures ranked by retention indices and fragmentation pattern from GC-EI-MS data sets. Over 20 PAH isomeric and deuterated standards were utilized for the GC-APLI-TIMS-TOF MS workflow validation. Noteworthy is the analytical capability for untargeted screening of isomeric and isobaric compounds with additional characterization metrics not available in traditional GC-EI-MSn workflows.

The juvenile hormone described in Rhodnius prolixus by Wigglesworth is juvenile hormone III skipped bisepoxide.

Juvenile hormones (JHs) are sesquiterpenoids synthesized by the corpora allata (CA). They play critical roles during insect development and reproduction. The first JH was described in 1934 as a "metamorphosis inhibitory hormone" in Rhodnius prolixus by Sir Vincent B. Wigglesworth. Remarkably, in spite of the importance of R. prolixus as vectors of Chagas disease and model organisms in insect physiology, the original JH that Wigglesworth described for the kissing-bug R. prolixus remained unidentified. We employed liquid chromatography mass spectrometry to search for the JH homologs present in the hemolymph of fourth instar nymphs of R. prolixus. Wigglesworth's original JH is the JH III skipped bisepoxide (JHSB3), a homolog identified in other heteropteran species. Changes in the titer of JHSB3 were studied during the 10-day long molting cycle of 4th instar nymph, between a blood meal and the ecdysis to 5th instar. In addition we measured the changes of mRNA levels in the CA for the 13 enzymes of the JH biosynthetic pathway during the molting cycle of 4th instar. Almost 90 years after the first descriptions of the role of JH in insects, this study finally reveals that the specific JH homolog responsible for Wigglesworth's original observations is JHSB3.

Common structural features facilitate the simultaneous identification and quantification of the five most common juvenile hormones by liquid chromatography-tandem mass spectrometry.

This study reports the development and application of a liquid chromatography method coupled to electrospray tandem mass spectrometry (LC-MS/MS) for the identification and quantification of the five most common juvenile hormone (JH) homologs and methyl farnesoate (MF). The protocol allows the simultaneous analysis in a single LC run of JH I, JH II, JH III, JH III bisepoxide (JHB3) and JH III skipped bisepoxide (JHSB3). The identification of JHs is based on multiple reaction monitoring (MRM), using two of the most abundant fragmentation transitions for each hormone. Addition of deuterated JH III as an internal standard permits the absolute quantification of the different JHs. The JH homologs common structural features led to similar chromatographic behavior, as well as related fragmentation patterns, which facilitated the simultaneous detection of all the homologs in a single LC-MS/MS run. The protocol detects JHs in the low femtomole range, allowing often the analysis of JH in individual insects. Fragmentation of each of the JH homologs generates unique diagnostic ions that permitted the identification and quantification of JHs from samples of different species of Diptera, Lepidoptera, Heteroptera and Hymenoptera. Having a simple protocol, which can undisputedly determine the identity of the homologs present in a particular species, provides us with the opportunity to identify and quantify JHs existing in insects that are pests, vector of diseases or important research models.

Three Dimensional Secondary Ion Mass Spectrometry Imaging (3D-SIMS) of Aedes aegypti ovarian follicles.

The mobilization of nutrient reserves into the ovaries of Aedes aegypti mosquitoes after sugar-feeding plays a vital role in the female's reproductive maturation. In the present work, three-dimensional secondary ion mass spectrometry imaging (3D-SIMS) was used to generate ultrahigh spatial resolution (~1 µm) chemical maps and study the composition and spatial distribution of lipids at the single ovarian follicle level (~100 µm in size). 3D-Mass Spectrometry Imaging (3D-MSI) allowed the identification of cellular types in the follicle (oocyte, nurse and follicular cells) using endogenous markers, and revealed that most of the triacyglycerides (TGs) were compartmentalized in the oocyte region. By comparing follicles from water-fed and sugar-fed females (n=2), 3D-MSI-Time of Flight-SIMS showed that TGs were more abundant in ovarian follicles of sugar-fed females; despite relative sample reproducibility per feeding condition, more biological replicates will better support the trends observed. While the current 3D-MSI-TOF-SIMS does not permit MS/MS analysis of the lipid species, complementary LC-MS/MS analysis of the ovarian follicles aided tentative lipid assignments of the SIMS data. The combination of these MS approaches is giving us a first glimpse of the distribution of functionally relevant ovarian lipid molecules at the cellular level. These new tools can be used to investigate the roles of different lipids on follicle fitness and overall mosquito reproductive output.

Inhibition of juvenile hormone synthesis in mosquitoes by the methylation inhibitor 3-deazaneplanocin A (DZNep).

Juvenile hormone (JH), synthesized by the corpora allata (CA), controls development and reproduction in mosquitoes through its action on thousands of JH-responsive genes. These JH-dependent processes can be studied using tools that increase or decrease JH titers in vitro and in vivo. Juvenile hormone acid methyl transferase (JHAMT) is a critical JH biosynthetic enzyme. JHAMT utilizes the methyl donor S-adenosyl-methionine (SAM) to methylate farnesoic acid (FA) into methyl farnesoate (MF), releasing the product S-adenosyl-L-homocysteine (AdoHcy), which inhibits JHAMT. S-adenosyl-homocysteine hydrolase (SAHH) catalyzes AdoHcy hydrolysis to adenosine and homocysteine, alleviating AdoHcy inhibition of JHAMT. 3-deazaneplanocin A (DZNep), an analog of adenosine, is an inhibitor of SAHH, and an epigenetic drug for cancer therapy. We tested the effect of DZNep on in vitro JH synthesis by CA of mosquitoes. DZNep inhibited JH synthesis in a dose-response fashion. Addition of MF, but not of FA relieved the inhibition, demonstrating a direct effect on JHAMT. In vivo experiments, with addition of DZNep to the sugar ingested by mosquitoes, resulted in a dose-response decrease in JH synthesis and JH hemolymphatic titers, as well as expression of early trypsin, a JH-dependent gene. Our studies suggest that DZNep can be employed to lower JH synthesis and titer in experiments evaluating JH-controlled processes in mosquitoes.

Effective Liquid Chromatography-Trapped Ion Mobility Spectrometry-Mass Spectrometry Separation of Isomeric Lipid Species.

Lipids are a major class of molecules that play key roles in different biological processes. Understanding their biological roles and mechanisms remains analytically challenging due to their high isomeric content (e.g., varying acyl chain positions and/or double bond locations/geometries) in eukaryotic cells. In the present work, a combination of liquid chromatography (LC) followed by high resolution trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was used to investigate common isomeric glycerophosphocholine (PC) and diacylglycerol (DG) lipid species from human plasma. The LC dimension was effective for the separation of isomeric lipid species presenting distinct double bond locations or geometries but was not able to differentiate lipid isomers with distinct acyl chain positions. High resolution TIMS-MS resulted in the identification of lipid isomers that differ in the double bond locations/geometries as well as in the position of the acyl chain with resolving power ( R) up to ∼410 ( R ∼ 320 needed on average). Extremely small structural differences exhibiting collision cross sections (CCS) of less than 1% (down to 0.2%) are sufficient for the discrimination of the isomeric lipid species using TIMS-MS. The same level of performance was maintained in the complex biological mixture for the biologically relevant PC 16:0/18:1 lipid isomers. These results suggest several advantages of using complementary LC-TIMS-MS separations for regular lipidomic analysis, with the main emphasis in the elucidation of isomer-specific lipid biological activities.

Juvenile hormone controls ovarian development in female Anopheles albimanus mosquitoes.

Anophelinae mosquitoes are vectors of human malaria, a disease that infects hundreds of millions of people and causes almost 600,000 fatalities annually. Despite their medical importance, laboratory studies on key aspects of Anophelinae reproductive biology have been limited, and in particular, relatively little is known about the role of juvenile hormone (JH) in the control of female reproduction. The study presented here attempts to fill a gap of knowledge in our understanding of the JH control of ovarian development in female Anophelinae mosquitoes, using Anopheles albimanus as a model. Our studies revealed that JH controls the tempo of maturation of primary follicles in An. albimanus in a similar manner to that previously described in Aedes aegypti. At adult eclosion JH hemolymph titer was low, increased in 1-day old sugar-fed insects, and decreased in blood fed individuals. JH titers decreased if An. albimanus females were starved, and were reduced if insects emerged with low teneral reserves, precluding previtellogenic ovarian development. However, absolute hemolymph titers were lower than Ae. aegypti. Decapitation experiments suggested that if teneral reserves are sufficient, factors from the head activate JH synthesis by the corpora allata (CA) during the first 9-12 h after adult emergence. In conclusion, our studies support the hypothesis that JH controls previtellogenic ovarian development in female An. albimanus mosquitoes, in a similar manner that have been described in Culicinae.

Discovery and targeted monitoring of polychlorinated biphenyl metabolites in blood plasma using LC-TIMS-TOF MS.

In the present work, the potential for rapid, targeted analysis of hydroxylated metabolites of polychlorinated biphenyls (OH-PCBs) in diluted human blood plasma using liquid chromatography coupled with trapped ion mobility spectrometry and TOF high resolution mass spectrometry (LC-TIMS-TOF MS) was evaluated. Experimental OH-PCB collisional cross section (CCSN2) and gas-phase candidate structures (<3% error) are reported for the first time and used, in addition to the LC retention time and accurate m/z, as OH-PCB identification features in order to increase the detection selectivity. The proposed LC-TIMS-TOF MS workflow combines a "dilute-and-shoot" sample preparation strategy, a robust liquid chromatography step, a high-resolving power mobility separation (R ~ 150-250) and high-resolution mass spectrometry (R ~ 30-40k) for the separation, identification and quantification of common OH-PCB isomers with limits of detection comparable to traditional workflows (e.g., LOD and LOQ of ~10 pg/mL and ~50 pg/mL, respectively). The higher selectivity and low detection limits provides multiple advantages compared to current methodologies that typically require long, labor-intensive preparation and/or derivatization steps prior to gas or liquid chromatography-mass spectrometry.

Coupling trapped ion mobility spectrometry to mass spectrometry: trapped ion mobility spectrometry-time-of-flight mass spectrometry versus trapped ion mobility spectrometry-Fourier transform ion cyclotron resonance mass spectrometry.

RATIONALE: There is a need for fast, post-ionization separation during the analysis of complex mixtures. In this study, we evaluate the use of a high-resolution mobility analyzer with high-resolution and ultrahigh-resolution mass spectrometry for unsupervised molecular feature detection. Goals include the study of the reproducibility of trapped ion mobility spectrometry (TIMS) across platforms, applicability range, and potential challenges during routine analysis. METHODS: A TIMS analyzer was coupled to time-of-flight mass spectrometry (TOF MS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) instruments for the analysis of singly charged species in the m/z 150-800 range of a complex mixture (Suwannee River Fulvic Acid Standard). Molecular features were detected using an unsupervised algorithm based on chemical formula and IMS profiles. RESULTS: TIMS-TOF MS and TIMS-FT-ICR MS analysis provided 4950 and 7760 m/z signals, 1430 and 3050 formulas using the general Cx Hy N0-3 O0-19 S0-1 composition, and 7600 and 22 350 [m/z; chemical formula; K; CCS] features, respectively. CONCLUSIONS: TIMS coupled to TOF MS and FT-ICR MS showed similar performance and high reproducibility. For the analysis of complex mixtures, both platforms were able to capture the major trends and characteristics; however, as the chemical complexity at the level of nominal mass increases with m/z (m/z >300-350), only TIMS-FT-ICR MS was able to report the lower abundance compositional trends.

JH biosynthesis and hemolymph titers in adult male Aedes aegypti mosquitoes.

Juvenile hormone (JH) is a major hormonal regulator in insects. In Aedes aegypti females, JH signals the completion of the ecdysis to the adult stage and initiates reproductive processes. Although the regulation of JH synthesis and titer in Ae. aegypti females has been extensively studied, relatively little is known about changes of JH synthesis and titers in male mosquitoes, as well as on the roles of JH controlling male reproductive biology. A better understanding of male mosquito reproductive biology, including an improved knowledge of the hormonal control of reproduction, could increase the likelihood of success of male-targeting vector control programs. Using a high performance liquid chromatography coupled to electrospray tandem mass spectrometry method, we measured JH biosynthesis and hemolymph levels in male mosquitoes during pupal and adult stages. Our results revealed tightly concomitant changes in JH biosynthesis and JH hemolymph titers. Synthesis of JH III was very low in late pupae, significantly increased during the first 24 h after adult eclosion, and then remained relatively constant during the first six days after adult eclosion. Feeding high sugar diets resulted in an increase of JH synthesis and titers, and starvation significantly decreased JH synthesis, but this effect could be reversed by changing the males back to a high sugar diet. JH synthesis rates were similar in virgin and mated males, but hemolymph JH levels were different in well-nourished virgin and mated males. Starvation resulted in a significant reduction in insemination rates; with well-nourished males inseminating 2 times more females than water-fed. Giving a 20% sugar meal for 24 h to those mosquitoes that were previously starved for 6 days, caused a significant rise in insemination rates, restoring them to levels similar to those recorded for 20% fed males. These results suggest that nutrition plays a role on male fecundity, and this effect might be mediated by JH.

Analysis of isomeric opioids in urine using LC-TIMS-TOF MS.

In the present work, a fast separation, identification and quantification workflow based on liquid chromatography coupled to trapped ion mobility in tandem with mass spectrometry (LC-TIMS-MS) is described for the analysis of common isomeric drugs of abuse and their metabolites in human urine. In particular, the analytical performance of LC-TIMS-MS is shown for identification based on retention time, collision cross section and accurate mass for three sets of common isomeric opioids and their deuterated analogs in urine. The LC-TIMS-MS analysis provided limits of detection of 1.4-35.2 ng/mL with demonstrated linearity up to 500 ng/mL, enabling discovery and targeted monitoring (DTM) of opioids in urine, with high precision in retention times (RT) (< 0.3%), collision cross sections (CCS) (< 0.6%) and mass accuracy (< 1 ppm) across multiple measurements using external calibration. A good agreement was observed between theoretical and experimental CCS from candidate structures optimized at the DFT/B3LYP level. The need for complementary liquid and mobility separations prior to mass analysis is shown for the analysis of complex mixtures, with mobility resolving power of 80-130. The reproducibility and high speed of LC-TIMS-MS analysis provides a powerful platform for drug and metabolite screening in biological matrices with higher precision and confidence than traditional LC-multiple reaction monitoring (MRM) approaches.

Towards Discovery and Targeted Peptide Biomarker Detection Using nanoESI-TIMS-TOF MS.

In the present work, the potential of trapped ion mobility spectrometry coupled to TOF mass spectrometry (TIMS-TOF MS) for discovery and targeted monitoring of peptide biomarkers from human-in-mouse xenograft tumor tissue was evaluated. In particular, a TIMS-MS workflow was developed for the detection and quantification of peptide biomarkers using internal heavy analogs, taking advantage of the high mobility resolution (R = 150-250) prior to mass analysis. Five peptide biomarkers were separated, identified, and quantified using offline nanoESI-TIMS-CID-TOF MS; the results were in good agreement with measurements using a traditional LC-ESI-MS/MS proteomics workflow. The TIMS-TOF MS analysis permitted peptide biomarker detection based on accurate mobility, mass measurements, and high sequence coverage for concentrations in the 10-200 nM range, while simultaneously achieving discovery measurements of not initially targeted peptides as markers from the same proteins and, eventually, other proteins. Graphical Abstract ᅟ.

Fast, ultra-trace detection of juvenile hormone III from mosquitoes using mass spectrometry.

In the present work, a new protocol for fast separation and quantification of JH III from biological samples using liquid chromatography coupled to electrospray tandem mass spectrometry is described. In particular, the proposed protocol improves existing methodologies by combining a limited number of sample preparation steps with fast LC-MS/MS detection, providing lower limits of detection and demonstrated matrix effect control, together with high inter and intraday reproducibility. A limit of detection of 8pg/mL (0.32pg on column) was achieved, representing a 15-fold gain in sensitivity with respect to previous LC-MS based protocols. The performance of the LC-MS/MS protocol is comparable to previously described JH III quantitation protocol based on fluorescence detection, with the added advantage that quantification is independent of the availability of fluorescent tags that are often unavailable or show quite diverse responses on a batch-to-batch basis. Additionally, a detailed description of the JH III fragmentation pathway is provided for the first time, based on isolation of the molecular ion and their intermediate fragments using in-source MS/MS, MS/MS(n) and FT-ICR MS/MS measurements. The JH III workflow was evaluated as a function of developmental changes, sugar feeding and farnesoic acid stimulation in mosquitoes and can be applied to the detection of other juvenile hormones.

Rapid ultra-trace analysis of sucralose in multiple-origin aqueous samples by online solid-phase extraction coupled to high-resolution mass spectrometry.

Because of its widespread consumption and its persistence during wastewater treatment, the artificial sweetener sucralose has gained considerable interest as a proxy to detect wastewater intrusion into usable water resources. The molecular resilience of this compound dictates that coastal and oceanic waters are the final recipient of this compound with unknown effects on ecosystems. Furthermore, no suitable methodologies have been reported for routine, ultra-trace detection of sucralose in seawater as the sensitivity of traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is limited by a low yield of product ions upon collision-induced dissociation (CID). In this work, we report the development and field test of an alternative analysis tool for sucralose in environmental waters, with enough sensitivity for the proper quantitation and confirmation of this analyte in seawater. The methodology is based on automated online solid-phase extraction (SPE) and high-resolving-power orbitrap MS detection. Operating in full scan (no CID), detection of the unique isotopic pattern (100:96:31 for [M-H](-), [M-H+2](-), and [M-H+4](-), respectively) was used for ultra-trace quantitation and analyte identification. The method offers fast analysis (14 min per run) and low sample consumption (10 mL per sample) with method detection and confirmation limits (MDLs and MCLs) of 1.4 and 5.7 ng/L in seawater, respectively. The methodology involves low operating costs due to virtually no sample preparation steps or consumables. As an application example, samples were collected from 17 oceanic and estuarine sites in Broward County, FL, with varying salinity (6-40 PSU). Samples included the ocean outfall of the Southern Regional Wastewater Treatment Plant (WWTP) that serves Hollywood, FL. Sucralose was detected above MCL in 78% of the samples at concentrations ranging from 8 to 148 ng/L, with the exception of the WWTP ocean outfall (at pipe end, 28 m below the surface) where the measured concentration was 8418 ± 3813 ng/L. These results demonstrate the applicability of this monitoring tool for the trace-level detection of this wastewater marker in very dilute environmental waters.

Analysis of drugs of abuse by online SPE-LC high resolution mass spectrometry: communal assessment of consumption.

An online SPE-LC-HRMS method was developed to monitor the consumption of 18 drugs of abuse (DOAs) including amphetamines, opioids, cocainics, cannabinoids, lysergics, and their corresponding metabolites in a well characterized college campus setting via wastewater analysis. Filtered and diluted (10×) sewage water samples (5 mL inj.) were automatically pre-concentrated and analyzed in 15 min using a Thermo EQuan MAX online SPE system equipped with a HyperSep™ Retain PEP (20×2.1 mm×12 µm) SPE column and a Hypersil Gold™ aQ (150×2.1 mm×3 µm) analytical column. A Q Exactive™ Hybrid Quadrupole-Orbitrap HRMS was used in full scan mode (R=140,000) for positive identification, and quantitation of target compounds. Method detection limits for all analytes ranged between 0.6 and 1.7 ng/L in sewage. A total of 14 DOAs were detected from two different locations (dorms and main college campus) within a one-year period. Most frequently detected drugs throughout the entire study were amphetamine (>96%) and THC's metabolite 11-nor-9-carboxy-Δ-9-THC (>100%) with maximum concentrations of 5956 and 2413 ng/L respectively. Daily doses per 1000 people were determined in order to assess consumption of THC, amphetamine, heroin and cocaine, in both dorms and main campus.

A simple method for routine monitoring of glyphosate and its main metabolite in surface waters using lyophilization and LC-FLD+MS/MS. Case study: canals with influence on Biscayne National Park.

A novel method was developed for the analysis of the herbicide glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) based on lyophilization. Sample preparation steps are limited to fortification with aspartic acid as internal standard and water removal by lyophilization (3-4 days for 72 samples), followed by suspension of dry residues in borate buffer (pH=9.0) and addition of ethylenediaminetetraacetic acid (EDTA) and 9-fluorenylmethylchloroformate (FMOC-Cl) for pre-column derivatization. The obtained derivatization mixture was injected on a highly endcapped C18 column where a basic pH gradient separation of the anionic analytes from neutral derivatization byproducts was achieved, with simultaneous quantitation by fluorescence and compound confirmation by tandem mass spectrometry. Method detection limits (for 20 mL samples) were 0.058 µg/L and 0.108 µg/L for glyphosate and AMPA, respectively. The method had a high dynamic range (0.1-50.0 µg/L) which allowed quantitation at both background and high levels of the herbicide. As a case study, the methodology was successfully applied to detect the occurrence of these compounds in water canals managed by the South Florida Water Management District. These canals will be used as freshwater source to hydrate estuarine wetlands of Biscayne National Park under the Comprehensive Everglades Restoration Project, in order to decrease ecosystem stress from hypersaline conditions caused by anthropogenic reduction of historical freshwater flow towards the Biscayne Bay. Method development, validation, advantages, limitations and measured environmental concentrations are discussed. This methodology has minimal requirements in terms of materials, instruments and analyst training, which could represent a desirable tool for laboratories interested in the monitoring of glyphosate in surface waters.