On-line Solid-phase Extraction-liquid Chromatography-tandem Research Articles

Determination of trace α-amanitin in urine of mushroom poisoning patient by online solid phase extraction-liquid chromatography-tandem mass spectrometry

An analytical method was established for the determination of trace α-amanitin in the urine of patients suffering from mushroom poisoning by online solid phase extraction-liquid chromatography-tandem mass spectrometry (online SPE-LC-MS/MS). The sample was protein precipitated with formic acid acidified acetonitrile-methanol (5:1, v/v). Reversed-phase liquid-liquid microextraction was used to remove the organic solvent from the sample extract. The toxin was purified by online SPE using an ODS micro column (5 mm×2.1 mm, 5 μm), and separated on an XBridgeTM BEH C18 column (150 mm×3.0 mm, 2.5 μm). Finally, the toxin was measured by MS/MS in the negative electrospray ionization (ESI-) mode. Multiple reaction monitoring (MRM) was used, and the conditions were m/z 917.4>205.1 (quantitative ion transition) and m/z 917.4>257.1. Collision energy for both transitions was 55 eV. A fast valve-switching technique with a quantitative loop was used as an interface between the online SPE and LC-MS/MS modules. The two modules were independent, neither the mobile phase nor the pressure would interfere with each other, thus ensuring the stability of the system. Precise purification by the online system could effectively eliminate the matrix effects in the subsequent MS detection. Weak matrix suppression effects were found, with results of 88.7%-96.5%. The linear range of α-amanitin in urine was 0.1-50 μg/L with a correlation coefficient (r2) of 0.9983. The limit of detection (LOD) and limit of quantification (LOQ) in the sample matrix were 0.03 μg/L and 0.1 μg/L, respectively. The average recoveries at three spiked levels (0.1, 2.0 and 20 μg/L) were 84.3%-91.7% with relative standard deviations (RSDs) of 3.8%-7.2%. The accuracy and precision were evaluated using quality control samples with toxin contents of 0.1 μg/L (LOQ), 0.2 μg/L (2-fold LOQ), 2.0 μg/L (medium level), and 20 μg/L (high level). The calculated average intra-day accuracy was 85.1%-96.0% with the precision of 4.1%-7.8%. The inter-day accuracy was 82.9%-94.8% with the precision of 5.0%-9.5%. The specificity of the method was verified by negative samples derived from patients who suffered only gastroenteritis poisoning, without hepatotoxic symptoms. α-Amanitin was found in urine samples from nine mushroom poisoning patients with hepatotoxic symptoms. The sampling time ranged from 19 h to 92 h. The toxin contents were 0.11-53.1 μg/L. For patients with a high intake of poisonous mushrooms, the toxin content was 53.1 μg/L in a patient's urine sampled 19 h after accidental consumption and 0.19 μg/L in another patient's urine sampled 92 h after poisoning. The content of α-amanitin was only 0.53 μg/L in the urine sample obtained 23 h after consumption for a patient with low intake and 0.11 μg/L in the urine sampled from another patient 40 h after poisoning. Amatoxins can metabolize rapidly in vivo. The laboratory identification of amatoxin poisoning requires a method for trace-level analysis in the biological matrix. It is proved that this method is simple, accurate and sensitive by the application to the analysis of actual samples. The protein precipitation and reversed-phase liquid-liquid microextraction steps are fast and simple. Hence, they can be used as a rapid and effective pre-treatment method for online SPE-LC-MS/MS analysis of water-soluble toxins in biomaterial matrix. Highly sensitive analysis of α-amanitin in urine can be obtained using a precise purification technology via online SPE in this study. The problem of qualitative confirmation of the toxin at trace levels (0.03 μg/L) after poisoning can be solved. The laboratory identification time for amatoxin poisoning in some patients exceeds 90 h. The developed analytical method at trace level (0.1 μg/L of LOQ) can provide reliable technical support for establishing the dose-response relationship of α-amanitin in vivo. It can satisfy for the determination of trace α-amanitin in urine samples from patients with hepatotoxic mushroom poisoning.

Online Turbulent Flow Extraction and Column Switching for the Confirmatory Analysis of Stimulants in Urine by Liquid Chromatography-Mass Spectrometry.

Stimulants are often used to treat attention deficit disorders and nasal congestion. As they can be misused and overdosed, the detection of stimulants is relevant in the toxicological field as well as in the doping control field. The effects of stimulants can indeed be beneficial for athletes. Therefore, their in-competition use is prohibited by the World Anti-Doping Agency (WADA). As stimulants represent one of the most detected categories of prohibited substances, automation of methods to detect and confirm their presence is desirable. Previous work has shown the advantages of using turbulent flow online solid-phase extraction liquid chromatography-tandem mass spectrometry (online SPE LC-MS-MS) for the detection and confirmation of diuretics and masking agents. Hence, a turbulent flow online SPE LC-MS-MS method, compliant with the WADA's identification criteria, was developed and validated for the detection and confirmation of 80 stimulants or metabolites with limits of identification varying between 10 (or possibly lower) and 100 ng/mL. As several metabolites are common metabolites for multiple administered stimulants, this means that with this method, misuse of well over 100 compounds can be detected. As the developed method uses the same columns and mobile phases as our turbulent flow online SPE LC-MS-MS method for the confirmation of diuretics and masking agents, there is no need to change the configuration of the instrument when switching between the diuretics method and the developed stimulants method.

Occurrence of Antibiotics in Influent and Effluent from 3 Major Wastewater-Treatment Plants in Finland.

Wastewater-treatment plants (WWTPs) are regarded as one of the main sources of antibiotics in the environment. In the present study, the concentrations of multiple antibiotics and their metabolites belonging to 5 antibiotic classes were determined in 3 major Finnish WWTPs. An online solid phase extraction-liquid chromatography-tandem mass spectrometry method was used for the extraction and analysis of the compounds. The method was fully validated using real and synthetic wastewaters. Seven antibiotics and 3 metabolites were found in the analyzed samples. Sulfonamides were removed most efficiently, whereas macrolides usually showed negative removal efficiency during the treatment, which means that the concentrations for individual antibiotics determined in the effluent samples were higher than in the influent samples. Sulfadiazine was found at concentrations up to 1018 ng/L, which was the highest concentration of any of the detected antibiotics in influent. In the effluent samples, the highest mean concentration was found for trimethoprim (532 ng/L). The measured mass loads of the antibiotics and metabolites to the receiving waters ranged from 2 to 157 mg/d per 1000 population equivalent. The evaluated environmental risk assessment showed that clarithromycin and erythromycin might pose a risk to the environment. The present study further underlines the importance of implementing technology for efficient removal of xenobiotics during wastewater treatment. Environ Toxicol Chem 2020;39:1774-1789. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A fully automated approach for the analysis of 37 psychoactive substances in raw wastewater based on on-line solid phase extraction-liquid chromatography-tandem mass spectrometry

This work presents a multi-residue method for the simultaneous determination of 37 legal and illicit psychoactive substances in wastewater, including the most common illicit drugs (cocaine-related compounds, amphetamine-type stimulants, hallucinogens, opiates/opioids, and cannabinoids), new psychoactive substances (two synthetic cathinones, the synthetic opioid AH-7921, and the arylcyclohexylamine methoxetamine), and legal but controlled psychoactive substances (stimulants, benzodiazepines, antidepressants, sedatives, antipsychotics, and hypnotics). To this end a fully automated analytical approach based on solid phase extraction coupled in series to liquid chromatography tandem mass spectrometry detection (on-line SPE- LC–MS/MS) was used. The methodology developed was validated in terms of linearity, recovery, repeatability, and sensitivity in wastewater. Method linearity was between 0.1 ng/L (or the analyte limit of quantification if higher) and 2,000 ng/L (10,250 ng/L in the case of caffeine). Absolute recoveries were variable (between 5% and 132%), depending on the analyte. However, the use of isotopically labeled compounds corrected for analyte losses during the extraction process and matrix effects (relative recoveries within the range of 80–120%). Repeatability of the method was satisfactory for all analytes, with RSD values lower than 13% for most compounds. Limits of detection and quantification in wastewater were below 7 and 23 ng/L, respectively, for all analytes except lormetazepam (10 and 32 ng/L), caffeine (13 and 44 ng/L), and the cannabinoids 11-nor-9-carboxy- Δ9-tetrahydrocannabinol (18 and 61 ng/L) and 11-hydroxy-Δ9- tetrahydrocannabinol (69 and 228 ng/L). The method was applied to the analysis of wastewater samples collected daily in Barcelona for one week. Twenty-five of the 37 analytes were detected in the samples analyzed. Average concentrations ranged from 7 ng/L in the case of zolpidem to 54 μg/L in the case of caffeine.

On-line solid phase extraction-liquid chromatography-tandem mass spectrometry for insect repellent residue analysis in surface waters using atmospheric pressure photoionization

Insect repellents (IRs) are a group of organic chemicals whose function is to prevent the ability of insects of landing in a surface. These compounds have been found in the environment and may pose a risk to non-target organisms. In this study, an on-line solid phase extraction – high performance liquid chromatography-tandem mass spectrometry multiresidue method was developed using an atmospheric photoionization source (SPE-HPLC-(APPI)-MS/MS). The use of the APPI as an alternative ionization technique to electrospray (ESI) and atmospheric pressure chemical ionization (APCI) allowed expanding the range of analytical techniques suitable for the analysis of IRs, so far relied in gas chromatography. High sensitivity and precision was reached with method limits of quantification between 0.2 and 4.6 ng l−1 and interday and intraday precision equal or below 15%. The validated method was applied to the study of surface water samples from three European river basins with different flow regime (Adige River in Italy, Sava River in the Balkans, and Evrotas River in Greece). The results showed that two IRs (DEET and Bayrepel) were ubiquitous in the Sava and Evrotas basins, reaching concentrations as high as 105 μg l−1 of Bayrepel in the Sava River, and 5 μg l−1 of DEET in the Evrotas River. Densely populated areas and effluent waste waters are pointed out as the responsible for this pollution. In the alpine river Adige, only three samples showed low levels of IRs (6.01–37.8 ng l−1). The concentrations measured were used to perform an environmental risk assessment based on the hazard quotients (HQs) estimation approach by using the chronic and acute eco-toxicity data available. The results revealed that despite the high frequency and eventually high concentrations of these IRs determined in the three basins, only few sites were at risk, with 1 < HQs < 3.3.

Development and Multi-laboratory Verification of US EPA Method 543 for the Analysis of Drinking Water Contaminants by Online Solid Phase Extraction-LC-MS-MS.

A drinking water method for seven pesticides and pesticide degradates is presented that addresses the occurrence monitoring needs of the US Environmental Protection Agency (EPA) for a future Unregulated Contaminant Monitoring Regulation (UCMR). The method employs online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS-MS). Online SPE-LC-MS-MS has the potential to offer cost-effective, faster, more sensitive and more rugged methods than the traditional offline SPE approach due to complete automation of the SPE process, as well as seamless integration with the LC-MS-MS system. The method uses 2-chloroacetamide, ascorbic acid and Trizma to preserve the drinking water samples for up to 28 days. The mean recoveries in drinking water (from a surface water source) fortified with method analytes are 87.1-112% with relative standard deviations of <14%. Single laboratory lowest concentration minimum reporting levels of 0.27-1.7 ng/L are demonstrated with this methodology. Multi-laboratory data are presented that demonstrate method ruggedness and transferability. The final method meets all of the EPA's UCMR survey requirements for sample collection and storage, precision, accuracy, and sensitivity.

Sensitive, automatic method for the determination of diazepam and its five metabolites in human oral fluid by online solid-phase extraction and liquid chromatography with tandem mass spectrometry.

A novel and simple online solid-phase extraction liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of diazepam and its five metabolites including nordazepam, oxazepam, temazepam, oxazepam glucuronide, and temazepam glucuronide in human oral fluid. Human oral fluid was obtained using the Salivette(®) collection device, and 100 μL of oral fluid samples were loaded onto HySphere Resin GP cartridge for extraction. Analytes were separated on a Waters Xterra C18 column and quantified by liquid chromatography with tandem mass spectrometry using the multiple reaction monitoring mode. The whole procedure was automatic, and the total run time was 21 min. The limit of detection was in the range of 0.05-0.1 ng/mL for all analytes. The linearity ranged from 0.25 to 250 ng/mL for oxazepam, and 0.1 to 100 ng/mL for the other five analytes. Intraday and interday precision for all analytes was 0.6-12.8 and 1.0-9.2%, respectively. Accuracy ranged from 95.6 to 114.7%. Method recoveries were in the range of 65.1-80.8%. This method was fully automated, simple, and sensitive. Authentic oral fluid samples collected from two volunteers after consuming a single oral dose of 10 mg diazepam were analyzed to demonstrate the applicability of this method.

An on-line solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of perfluoroalkyl acids in drinking and surface waters.

An UHPLC-MS/MS multiresidue method based on an on-line solid phase extraction (SPE) procedure was developed for the simultaneous determination of 9 perfluorinated carboxylates (from 4 to 12 carbon atoms) and 3 perfluorinated sulphonates (from 4 to 8 carbon atoms). This work proposes using an on-line solid phase extraction before chromatographic separation and analysis to replace traditional methods of off-line SPE before direct injection to LC-MS/MS. Manual sample preparation was reduced to sample centrifugation and acidification, thus eliminating several procedural errors and significantly reducing time-consuming and costs. Ionization suppression between target perfluorinated analytes and their coeluting SIL-IS were detected for homologues with a number of carbon atoms less than 9, but the quantitation was not affected. Total matrix effect corrected by SIL-IS, inclusive of extraction efficacy, and of ionization efficiency, ranged between −34 and +39%. The percentage of recoveries, between 76 and 134%, calculated in different matrices (tap water and rivers impacted by different pollutions) was generally satisfactory. LODs and LOQs of this on-line SPE method, which also incorporate recovery losses, ranged from 0.2 to 5.0 ng/L and from 1 to 20 ng/L, respectively. Validated on-line SPE-LC/MS/MS method has been applied in a wide survey for the determination of perfluoroalkyl acids in Italian surface and ground waters.

Quantification of nerve agent biomarkers in human serum and urine.

A novel method for rapid and sensitive quantification of the nerve agent metabolites ethyl, isopropyl, isobutyl, cyclohexyl, and pinacolyl methylphosphonic acid has been established by combining salting-out assisted liquid-liquid extraction (SALLE) and online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS). The procedure allows confirmation of nerve agent exposure within 30 min from receiving a sample, with very low detection limits for the biomarkers of 0.04-0.12 ng/mL. Sample preparation by SALLE was performed in less than 10 min, with a common procedure for both serum and urine. Analyte recoveries of 70-100% were obtained using tetrahydrofuran as extraction solvent and Na2SO4 to achieve phase separation. After SALLE, selective analyte retention was obtained on a ZrO2 column by Lewis acid-base and hydrophilic interactions with acetonitrile/1% CH3COOH (82/18) as the loading mobile phase. The phosphonic acids were backflush-desorbed onto a polymeric zwitterionic column at pH 9.8 and separated by hydrophilic interaction liquid chromatography. The method was linear (R(2) ≥ 0.995) from the limits of quantification to 50 ng/mL, and the within- and between-assay repeatability at 20 ng/mL were below 5% and 10% relative standard deviation, respectively.

Design of online solid phase extraction‐liquid chromatography‐tandem mass spectrometry (SPE‐LC‐MS/MS) hyphenated systems for quantitative analysis of small organic compounds in biological matrices

Three online solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method examples are presented where two different types of chromatographic columns or solvent systems were coupled to meet specific analytical objectives: (i) SPE of target analytes by restricted access media from high ionic strength urine matrix was coupled with reversed phase LC-MS/MS conditions accommodating high ionization potentials of the analytes (urinary bisphenol A and other phenolic derivatives); (ii) strong cation exchange SPE of analytes of diverse polarity and pK(a) was coupled with reversed phase LC-MS/MS analysis (urinary atrazine metabolites); (iii) pre-concentration of low pg per sample analytes by weak anion exchange SPE was hyphenated with ion pair LC-MS analysis (intracellular nucleotide triphosphate analogs). With these examples we suggest a conductive generic work flow for the development of online SPE-LC-MS methods and show how advanced commercial LC devices and software allow for the design of complex yet highly versatile analytical separation systems suited to the unique physicochemical properties of the target analytes.

High-Throughput Simultaneous Analysis of Five Urinary Metabolites of Areca Nut and Tobacco Alkaloids by Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry with On-Line Solid-Phase Extraction

Areca nut and tobacco are commonly used drugs worldwide and have been frequently used in combination. We describe the use of on-line solid-phase extraction and isotope-dilution liquid chromatography-tandem mass spectrometry for the simultaneous measurement of five major urinary metabolites of both areca nut and tobacco alkaloids, namely, arecoline, arecaidine, N-methylnipecotic acid, nicotine, and cotinine. Automated purification of urine was accomplished with a column-switching device. After the addition of deuterium-labeled internal standards, urine samples were directly analyzed within 13 minutes. This method was applied to measure urinary metabolites in 90 healthy subjects to assess areca nut/tobacco exposure. Urinary time course of arecoline, arecaidine, and N-methylnipecotic acid was investigated in five healthy nonchewers after oral administration of areca nut water extracts. The limits of detection were 0.016 to 0.553 ng/mL. Interday and intraday imprecision were <10%. Mean recoveries of five metabolites in urine were 97% to 114%. Mean urinary concentrations of arecoline, arecaidine, N-methylnipecotic acid, nicotine, and cotinine in regular areca nut chewers also smokers were 23.9, 5,816, 1,298, 2,635, and 1,406 ng/mg creatinine, respectively. Time course study revealed that after administration of areca nuts extracts, the major urinary metabolite was arecaidine with a half-life of 4.3 hours, followed by N-methylnipecotic acid with a half-life of 7.9 hours, and very low levels of arecoline with a half-life of 0.97 hour. This on-line solid-phase extraction liquid chromatography-tandem mass spectrometry method firstly provides high-throughput direct analysis of five urinary metabolites of areca nut/tobacco alkaloids. This method may facilitate the research into the oncogenic effects of areca nut/tobacco exposure.

Risk assessment of representative and priority pesticides, in surface water of the Alqueva reservoir (South of Portugal) using on-line solid phase extraction-liquid chromatography-tandem mass spectrometry

Surface waters located in intensive agricultural areas are more vulnerable to the pesticides contamination, which is a major concern if the water is intended to be used for human consumption. The aim of this study was to evaluate the presence and the distribution of pesticides in the Alqueva reservoir, an important source of water supply (South of Portugal), considering their representativeness in the agricultural practice of the area. For the analysis of pesticides risk impact we used the environmental quality standards in the field of water policy proposed recently by the European Commission. The pesticides belonging to the classes of phenylureas, triazines, chloroacetanilides, organophosphorous and thiocarbamates were analysed by on-line solid phase extraction-liquid chromatography-tandem mass spectrometry. The pesticides more frequently detected were atrazine, simazine, diuron and terbuthylazine. The highest levels of these pesticides were registered in spring, after pesticides treatment, namely in olive-tree and vine crops. The priority pesticides atrazine and diuron reached values above the annual average proposed in the European Union Legislation. The herbicide atrazine reached values that surpassed the proposed maximum allowable concentration (2 000 ng L − 1 ). The sampling stations most affected by these pesticides were Sra. Ajuda, Lucefecit and Alcarrache, located in the northern part of the reservoir, closer to Spain where the agricultural activity is more intensive.