Cesar E. Ramirez

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 THCs 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.

Stability of dioctyl sulfosuccinate (DOSS) towards hydrolysis and photodegradation under simulated solar conditions

Dioctyl sulfosuccinate (DOSS) is one of the main components of Corexit® EC9500A, a chemical dispersant formulation used at the surface and at depth during the response to the Deepwater Horizon incident. Despite being a high volume use chemical, data on its environmental stability are scarce. Hydrolysis and photodegradation of DOSS in both pure water and seawater were reported in the present study. DOSS photodegraded much faster under ultraviolet light source (254 nm, with half-life in hours) compared to relevant environmental light sources i.e., 350 nm and solar simulator (with half-lives in days). LC/MS-MS analysis of hydrolysis and photo-irradiated samples showed the presence of a common degradation product. MS/MS fragmentation of that product indicated a substitution of an octyl group by a hydroxyl group with a corresponding formula of C12H21O7S, which was confirmed by HRMS detection (Q-TOF, m/z 309.1017, +1.29 ppm).

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.

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.

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

AbstractIn 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 Abstractᅟ

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.

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.