High-performance liquid chromatography (HPLC) and high-performance liquid chromatography mass spectrometry (HPLC/MS) for the analysis of date rape drugs

The drugs studied in this work have been reportedly used to commit drug-facilitated sexual assault (DFSA), commonly known as "date rape". Detection of the drugs was performed using high-performance liquid chromatography with ultraviolet detection (HPLC/UV) and identified with high performance-liquid chromatography mass spectrometry (HPLC/MS) using selected ion monitoring (SIM). The objective of this study was to develop a single HPLC method for the simultaneous detection, identification and quantitation of these drugs. The following drugs were simultaneously analyzed: Gamma-hydroxybutyrate (GHB), scopolamine, lysergic acid diethylamide, ketamine, flunitrazepam, and diphenhydramine. The results showed increased sensitivity with electrospray (ES) ionization versus atmospheric pressure chemical ionization (APCI) using HPLC/MS. HPLC/ES/MS was approximately six times more sensitive than HPLC/APCI/MS and about fifty times more sensitive than HPLC/UV. A limit of detection (LOD) of 100 ppb was achieved for drug analysis using this method. The average linear regression coefficient of correlation squared (r2) was 0.933 for HPLC/UV and 0.998 for HPLC/ES/MS. The detection limits achieved by this method allowed for the detection of drug dosages used in beverage tampering. This method can be used to screen beverages suspected of drug tampering. The results of this study demonstrated that solid phase microextraction (SPME) did not improve sensitivity as an extraction technique when compared to direct injections of the drug standards.

Assessment of the occurrence and potential risks of antibiotics and their metabolites in South Florida waters using liquid chromatography tandem mass spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. ^ The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can't be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications.^ Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. ^ Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.^

Assessment of the Occurrence and Potential Risks of Antibiotics and their Metabolites in South Florida Waters Using Liquid Chromatography Tandem Mass Spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can’t be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications. Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.

The effects of self-monitoring on homework completion and accuracy rates of students with disabilities in an inclusive general education classroom

This study investigated the effects of self-monitoring on the homework completion and accuracy rates of four, fourth-grade students with disabilities in an inclusive general education classroom. A multiple baseline across subjects design was utilized to examine four dependent variables: completion of spelling homework, accuracy of spelling homework, completion of math homework, accuracy of math homework. Data were collected and analyzed during baseline, three phases of intervention, and maintenance. ^ Throughout baseline and all phases, participants followed typical classroom procedures, brought their homework to school each day and gave it to the general education teacher. During Phase I of the intervention, participants self-monitored with a daily sheet at home and on the computer at school in the morning using KidTools (Fitzgerald & Koury, 2003); a student friendly, self-monitoring program. They also participated in brief daily conferences to review their self-monitoring sheets with the investigator, their special education teacher. Phase II followed the same steps except conferencing was reduced to two days a week, which were randomly selected by the researcher and Phase III conferencing was one random day a week. Maintenance data were taken over a two-to-three week period subsequent to the end of the intervention. ^ Results of this study demonstrated self-monitoring substantially improved spelling and math homework completion and accuracy rates of students with disabilities in an inclusive, general education classroom. On average, completion and accuracy rates were highest over baseline in Phase III. Self-monitoring led to higher percentages of completion and accuracy during each phase of the intervention compared to baseline, group percentages also rose slightly during maintenance. Therefore, results suggest self-monitoring leads to short-term maintenance in spelling and math homework completion and accuracy. ^ This study adds to the existing literature by investigating the effects of self-monitoring of homework for students with disabilities included in general education classrooms. Future research should consider selecting participants with other demographic characteristics, using peers for conferencing instead of the teacher, and the use of self-monitoring with other academic subjects (e.g., science, history). Additionally, future research could investigate the effects of each of the two self-monitoring components used alone, with or without the conferencing.^