Phase Extraction Liquid Chromatography-tandem Mass Research Articles

Toxicity of 15 veterinary pharmaceuticals in zebrafish (Danio rerio) embryos

Extensive use of veterinary pharmaceuticals may result in contamination of water bodies adjacent to pasture land or areas where animal manure has been applied. In order to evaluate the potential risk to fish embryos 15 veterinary pharmaceuticals were investigated by use of an extended zebrafish embryo toxicity test. Chemical analysis of the exposure medium was performed by solid phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) for 11 of the compounds and potential metabolism by the embryos was studied for albendazole, febantel, fenbendazole and oxfendazole. Newly fertilized zebrafish eggs were exposed under static conditions in 96-well plates for 6 days to the pharmaceuticals: 5 antibacterials and 10 antiparasitics. Endpoints including mortality, malformations and other sublethal responses were recorded at 24, 48 and 144h post fertilization (hpf). The pharmaceuticals causing the highest toxicity were antiparasitics whereas the tested antibacterials, danofloxacin, enrofloxacin, tylosine, trimethoprim and oxytetracyclin had a much lower toxic potency in zebrafish embryos. Most toxic were fenbendazole, albendazole and flumethrin with no observed effect concentrations (NOECs) around 0.02mg/L. The overall NOEC was determined by lethality for the following pharmaceuticals: albendazole, fenbendazole and oxfendazole. Sublethal endpoints, including malformations, side-laying embryos, tremors, reduced movements and altered heart rate increased the sensitivity of the tests and determined the overall NOECs for febantel, doramectin, ivermectin, flumethrin and toltrazuril. Exposure to doramectin and ivermectin caused a decrease in movements at 24hpf and a decrease in heart rate at 48hpf. Flumethrin exposure resulted in decreased time to hatching, except at the highest concentrations, and caused an increase in heart rate at 48hpf. In contrast, toltrazuril caused an increased time to hatching and a decrease in heart rate. Chemical analysis of the exposure medium after the tests revealed great differences between nominal and measured concentrations, emphasizing the need of including analysis of the actual exposure concentrations. The results indicated that metabolism of albendazole into its sulfoxide protected the embryos from toxicity. Albendazole was metabolized efficiently into albendazole sulfoxide at lower exposure concentrations, resulting in reduced toxicity. At higher concentrations, an increasing proportion of albendazole remained unmetabolized and embryo mortality occurred. Metabolism by the embryos of febantel into fenbendazole and oxfendazole and of fenbendazole into oxfendazole was demonstrated. It is suggested that the toxic effect of febantel in zebrafish embryos is due to metabolism into fenbendazole.

High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography–mass spectrometry

Oxysterols are important in numerous biological processes, including cell signaling. Here we present an automated filtration/filter backflush-solid phase extraction-liquid chromatography–tandem mass spectrometry (AFFL-SPE-LC–MS/MS) method for determining 24-hydroxysterol and the isomers 25-hydroxycholesterol and 22S-hydroxycholesterol that enables simplified sample preparation, high sensitivity (∼25pg/mL cell lysis sample) and low sample variability. Only one sample transfer step was required for the entire process of cell lysis, derivatization and determination of selected oxysterols. During the procedure, autoxidation of cholesterol, a potential/common problem using standard analytical methods, was found to be negligible. The reversed phase AFFL-SPE-LC–MS/MS method utilizing a 1mm inner diameter column was validated, and used to determine levels of the oxysterol analytes in mouse fibroblast cell lines SSh-LII and NIH-3T3, and human cancer cell lines, BxPC3, HCT-15 and HCT-116. In BxPC3 cells, the AFFL-SPE-LC–MS/MS method was used to detect significant differences in 24S-OHC levels between vimentin+ and vimentin− heterogenous sub-populations. The methodology also allowed monitoring of significant alterations in 24S-OHC levels upon delivery of the Hedgehog (Hh) antagonist MS-0022 in HCT-116 colorectal carcinoma cell lines.

Method for the determination of perfluorinated compounds (PFCs) in water by solid-phase extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS)

A solid phase extraction-liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS) analytical method for the analysis of perfluorinated compounds (PFCs) in water is presented. The method incorporates a stable isotope internal standard (IS) for each target analyte added prior to sample collection. Method validation for perfluorinated carboxylic acids (PFCAs) (C4–C12), perfluorinated sulfonic acids (PFSAs) (C4, C6, and C8), and perfluorooctane sulfonamide (PFOSA) in laboratory Milli Q water evaluated the following: (1) method accuracy and precision over the concentration range 0.025 ng mL−1 to 25 ng mL−1; (2) internal standard calibration; (3) quantitation of mixed branched and linear isomers of perfluorooctanoate (PFOA) and perfluorooctane sulfonate (PFOS) with linear isomer calibration; (4) method minimum reporting levels (MRLs) (Shoemaker et al, http://www.epa.gov/nerlcwww/Method%20537_FINAL_rev1.1.pdf, and Winslow et al, Environ. Sci. Technol., 2006, 40, 281–288); and (5) 28 day holding time at the MRL, low, and mid range of the calibration range. IS calibration with IS addition to the sample bottle prior to sample collection demonstrated an overall method accuracy of at least 100 ± 10% with a precision of ±15% and a 28-day sample holding time at the MRLs for all target analytes. Method application to groundwater, production facility non-contact cooling water, and production facility wastewater treatment effluent samples demonstrated a method accuracy of at least 100 ± 19% with a precision of ±25% for all target analytes.

High-throughput analysis of testosterone in serum samples by on-line solid phase extraction liquid chromatography-tandem mass spectrometry

Liquid chromatography tandem-mass spectrometer (LC-MS/MS) techniques are more and more common in the measurement of testosterone concentrations in biological samples. However, LC-MS/MS methods are more laborious than streamlined automated immunochemistry methods because of the need for tedious pre-purification of the sample before the mass spectrometric analysis. We therefore developed a robust and rapid sample clean-up method to improve the throughput of the whole LC-MS/MS analysis procedure by applying an automated on-line solid-phase extraction (SPE) technique instead of the still widely used conventional liquid-liquid extraction. Testosterone was purified by the on-line SPE-column-switching technique after rapid precipitation of serum samples by zinc sulphate/internal standard solution before LC-MS/MS analysis. The results were compared with those of our previous routine LC-MS/MS method using liquid-liquid extraction with tert-butyl methyl ether for the pre-purification of the samples. The tested on-line SPE-LC-MS/MS method reached the specifications of the previous method with liquid-liquid extraction. The precision of the new method was notably better, especially in the lower concentration range, than with the former method; the total variation was below 10% in the whole quantitation range of 0.25-35 nmol/L. The new method liberates more than 50% of hands-on time of laboratory technicians as well as expensive instrument time for other applications compared with the older method. The on-line SPE-pre-purification technique tested in long-term use offers a rapid and reliable technique in the LC-MS/MS analysis of serum testosterone and is a valuable tool in the improvement of efficiency in the laborious steroid analytics.

Development and validation of a high-throughput double solid phase extraction–liquid chromatography–tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL −1 for urine and 20 ng mL −1 for plasma. The limit of detection and limit of quantification were 0.13 ng mL −1 and 2.5 ng mL −1, respectively, for both biological matrices. Recoveries were in the range of 75–81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL −1, but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng μmol −1 creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.

Evaluation of drugs of abuse use and trends in a prison through wastewater analysis

Illicit drugs and metabolites have been recognized recently as a group of emerging contaminants of concern, as a consequence of their high volumes of use and production. Drug residue levels in the aquatic environment have also been pointed out as good indicators of illicit drug abuse. The present work assesses for the first time drug abuse in a penal complex from the levels of different drug residues measured in the prison sewage waters and evaluates the suitability of this approach to track and control illicit drug usage in such facilities. The presence of various drugs of abuse and metabolites in sewage waters from a penal complex was determined by an analytical method based on on-line solid phase extraction-liquid chromatography–tandem mass spectrometry. Levels of consumption indicators measured in this water were used to back-calculate drugs usage in the penal complex. Daily use was observed for methadone (average of 156 doses/day/1000 inh), alprazolam (129 doses/day/1000 inh), ephedrine (46 doses/day/1000 inh), cannabis (33 doses/day/1000 inh.) and cocaine (3 doses/day/1000 inh). Sporadic consumption was observed for heroin, amphetamine, methamphetamine, and ecstasy. In spite of the fact that this and other sewage epidemiological approaches described may suffer from bias that still need to be investigated and refined, it provides near “real-time” information on collective drug use in an anonymous way and constitutes a very useful, economic and fast tool to evaluate the efficiency of measures adopted to control and track drug abuse in this type of facilities (or any other provided that has a STP associated or an accessible collector system).

Validation of an off line solid phase extraction liquid chromatography–tandem mass spectrometry method for the determination of systemic insecticide residues in honey and pollen samples collected in apiaries from NW Spain

The use of pesticides to protect crops against plagues and insects is one of the most important ways to assure agricultural quality and productivity. However, bad application practices may cause the contamination of different environmental compartments and animal species, as a consequence of migration or accumulation of those compounds. Fipronil, imidacloprid and thiametoxam are systemic or systemic-like insecticides widely used in maize crops. Their heavy action in the nervous system of target insects also means a high toxicity to non-target pollinator insects such as honey bees which can get in touch with them through pollen and nectar during foraging activities. These insecticides have even been suspected to cause a significant decrease of honeybee colonies that has been observed in many countries since the past decade. Since September 1st 2008, the European Commission set new MRLs in food and feed of plant and animal origin. The pesticides included in this study have MRLs in honey and pollen between 10 and 50 ng g −1. In the present work, an analytical method was developed with the aim of determining residues of fipronil and some of its metabolites (fipronil sulfone, fipronil sulfide, fipronil desulfinyl and fipronil carboxamide), thiamethoxam and imidacloprid in honey and pollen samples. The extraction optimization was performed using a Doehlert experimental design by studying two factors, the mixture and the ratio of solvents used. Prior to the extraction procedure, raw hive samples containing honey, pollen and wax were centrifuged at 4000 rpm. The upper solid material was removed, and 1 g of the lower phase was mixed with 3 mL of the optimized mixture of methanol/water (10/90). The extract was passed through a florisil cartridge and the target compounds were eluted with methanol and analysed by LC–MS/MS in selective reaction monitoring (SRM) mode. The method was validated according to the guidelines included in the SANCO/10684/2009 document and the ISO 11843 standard for the following parameters: decision limit (CCα), detection capability (CCβ), recovery, repeatability and reproducibility at 0.5, 1 and 1.5 folds the MRLs. Ion suppression/enhancement effects into the ion source were also assessed. The CCβ values were included between 0.83 and 4.83 ng g −1, well below the current MRLs. The validated method was applied to the determination of the target pesticides in 91 samples collected in colonies from 73 apiaries of NW Spain (two sampling campaigns during 2008). None of the target insecticides were detected among all the collected samples.

Evaluation of two pilot scale membrane bioreactors for the elimination of selected surfactants from municipal wastewaters

Summary The removal of selected surfactants, linear alkylbenzene sulfonates (LAS), coconut diethanol amides (CDEA) and alkylphenol ethoxylates and their degradation products were investigated using a two membrane bioreactor (MBR) with hollow fiber and plate and frame membranes. The two pilot plants MBR run in parallel to a full-scale conventional activated sludge (CAS) treatment. A total of eight influent samples with the corresponding effluent samples were analysed by solid phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS–MS). The results indicate that both MBR have a better effluent quality in terms of chemical and biological oxygen demand (COD and BOD), NH 4 + , concentration and total suspended solids (TSS). MBR showed a better similar performance in the overall elimination of the total nonylphenolic compounds, achieving a 75% of elimination or a 65% (the same elimination reached by CAS). LAS and CDEA showed similar elimination in the three systems investigated and no significant differences were observed.

Quantitative measurement of propofol and in main glucuroconjugate metabolites in human plasma using solid phase extraction–liquid chromatography–tandem mass spectrometry

A high-performance liquid chromatographic method coupled with tandem mass spectrometry detection has been developed for the determination of propofol and its main glucuroconjugate metabolites (propofol–glucuronide (PG), 1-quinol-glucuronide (1-QG) and 4-quinol-glucuronide (4-QG) in human plasma. All compounds were extracted with a single solid phase extraction procedure using Max Oasis © cartridges. Propofol and thymol (internal standard) were analyzed using a C8 reversed-phase column with a mobile phase consisting of methanol–water (75:25, v/v) containing 0.025% NH 4OH. Chromatography of glucuroconjugate metabolites and phenyl-β- d-glucuronide (internal standard) was performed using a hydrophilic interaction liquid chromatography (HILIC) and a mixture of acetonitrile/water/ammonium acetate buffer (100 mM, pH 5, 87/1/12, v/v/v). Both chromatographic separations were achieved in isocratic mode allowing a rapid analysis without re-equilibration of the phase. The method is specific and sensitive with a range of 10–1500 ng mL −1 for propofol and 1-QG, 20–3000 ng mL −1 for PG and 25–3750 ng mL −1 for 4-QG. The regression curves were linear for all compounds. The method is accurate and precise with intra-assay and inter-assay precision <8% and bias ≤6% for all compounds. This assay has allowed the successful measurement of propofol and its main glucuroconjugate metabolites in human plasma from 24 patients undergoing anaesthesia for elective partial hepatectomy surgery.

Quantification and confirmation of anionic, cationic and neutral pesticides and transformation products in water by on-line solid phase extraction–liquid chromatography–tandem mass spectrometry

Two on-line SPE–LC–ESI–MS/MS methods have been developed for the rapid determination and confirmation of 18 polar pesticides and nine transformation products (TPs) in water samples. Given the very different physico-chemical characteristics of the analytes, it was not feasible the simultaneous determination of all selected compounds in only one method. Thus, it was necessary to use heptafluorobutyric acid and formic acid in order to obtain good retention in the SPE cartridge for basic and acidic analytes, respectively. The developed analytical methodology based on the direct injection of 2 mL of water sample in the system allowed the quantification of all analytes at the 25 ng/L level (LOQ) with limits of detection normally lower than 5 ng/L. Satisfactory recoveries (70–110%) were obtained for most compounds in ground and surface water samples. Some exceptions were found mainly in surface water, due to the ion suppression produced by the higher amount of matrix interferents in these samples. The acquisition of two MS/MS transitions for each compound allowed the reliable confirmation of positive findings even at the LOQ level. The developed methodology was applied to real ground and surface water samples showing the interest of including TPs in monitoring methods, as several of them were found at concentrations higher than that of parent compounds.

Fully automated online solid phase extraction coupled directly to liquid chromatography–tandem mass spectrometry: Quantification of sulfonamide antibiotics, neutral and acidic pesticides at low concentrations in surface waters

A fully automated online solid phase extraction–liquid chromatography–tandem mass spectrometry (SPE–LC–MS/MS) instrumental setup has been developed for the quantification of sulfonamide antibiotics and pesticides in natural water. The direct coupling of an online solid phase extraction cartridge (Oasis HLB) to LC–MS/MS was accomplished using column switching techniques. High sensitivity in the low ng/L range was achieved by large volume injections of 18 mL with a combination of a tri-directional autosampler and a dispenser system. This setup allowed high sample throughput with a minimum of investment costs. Special emphasis was placed on low cross contamination. The chosen approach is suitable for research as well as for monitoring applications. The flexible instrumental setup was successfully optimised for different important groups of bioactive chemicals resulting in three trace analytical methods for quantification of (i) sulfonamide antibiotics and their acetyl metabolites; (ii) neutral pesticides (triazines, phenylureas, amides, chloracetanilides) and (iii) acidic pesticides (phenoxyacetic acids and triketones). Absolute extraction recoveries from 85 to 112% were obtained for the different analytes. More than 500 samples could be analyzed with one extraction cartridge. The inter-day precision of the method was excellent indicated by relative standard deviations between 1 and 6%. High accuracy was achieved by the developed methods resulting in maximum deviation relative to the spiked amount of 8–15% for the different analytes. Detection limits for various environmental samples were between 0.5 and 5 ng/L. Matrix induced ion suppression was in general smaller than 25%. The performance of the online methods was demonstrated with measurements of concentration dynamics of sulfonamide antibiotics and pesticides concentrations in a little creek during rain fall events.

Generic solid phase extraction–liquid chromatography–tandem mass spectrometry method for fast determination of drugs in biological fluids

A generic method was developed for the fast determination of a wide range of drugs in serum or plasma. The methodology comprises generic solid-phase extraction, on-line coupled to gradient HPLC with tandem mass spectrometric detection (SPE–LC–MS/MS). The individual components of the SPE–LC–MS/MS system were optimized in an integrated approach to maximize the application range and minimize the method development time. The optimized generic SPE–LC–MS/MS protocol was evaluated for 11 drugs with different physicochemical properties. Good quantification for 10 out of 11 of the pharmaceuticals in serum or plasma could be readily achieved. The quantitative assays gave recoveries better than 95%, lower quantification limits of 0.2–2.0 ng/ml, acceptable precision and accuracy and good linearity over 2–4 orders of magnitude. Carry-over was determined to be in the range of 0.02–0.10%, without optimization.

An on-line solid phase extraction—Liquid chromatography—Tandem mass spectrometry method for the analysis of citalopram, fluvoxamine, and paroxetine in human plasma

A specific and sensitive direct-injection high performance liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS-MS) method has been developed for the rapid identification and quantitative determination of citalopram, fluvoxamine, and paroxetine in human plasma. After dilution with 0.1% formic acid, plasma samples were injected into the LC-MS-MS system. Proteins and other large biomolecules were removed during an on-line sample cleanup step. The inter- and intra-assay coefficients of variation for all compounds were <11%. The total analysis time was 6 min per sample. The proposed method permits direct analysis of plasma samples without time-consuming sample preparation.

Behavioral states and the central nervous system

Used frequently in food contact materials, bisphenol A (BPA) has been studied extensively in recent years, and ubiquitous exposure in the general population has been demonstrated worldwide. Characterizing within- and between-individual variability of BPA concentrations is important for characterizing exposure in biomonitoring studies, and this has been investigated previously in adults, but not in children. The aim of this study was to characterize the short-term variability of BPA in spot urine samples in young children. Children aged ≥ 2–< 4 years (n = 25) were recruited from an existing cohort in Queensland, Australia, and donated four spot urine samples each over a two day period. Samples were analysed for total BPA using isotope dilution online solid phase extraction–liquid chromatography–tandem mass spectrometry, and concentrations ranged from 0.53 to 74.5 ng/ml, with geometric mean and standard deviation of 2.70 ng/ml and 2.94 ng/ml, respectively. Sex and time of sample collection were not significant predictors of BPA concentration. The between-individual variability was approximately equal to the within-individual variability (ICC = 0.51), and this ICC is somewhat higher than previously reported literature values. This may be the result of physiological or behavioural differences between children and adults or of the relatively short exposure window assessed. Using a bootstrapping methodology, a single sample resulted in correct tertile classification approximately 70% of the time. This study suggests that single spot samples obtained from young children provide a reliable characterization of absolute and relative exposure over the short time window studied, but this may not hold true over longer timeframes.