Determination of seven drugs of abuse and their metabolites in surface and wastewater using solid‐phase extraction coupled to liquid chromatography with high‐resolution mass spectrometry

A method based on liquid chromatography with electrospray ionization high‐resolution mass spectrometry (Exactive Orbitrap) combined with solid‐phase extraction using a strong cationic exchange mixed‐mode sorbent has been developed for the determination of seven drugs of abuse, including two synthetic cathinones, as well as some of their metabolites in environmental water samples. The method provides low detection limits and a high confirmation power thanks to the diagnostic and two fragment ions monitored for each compound in high‐resolution mass spectrometry, providing six identification points for each analyte. The clean‐up step based on methanol in the extraction step adequately decreased the matrix effect, mainly for river and effluent water, and provided suitable process efficiency. Method detection and quantitation limits for environmental waters were at low nanogram per liter. The method was applied to analyze the samples of influent and effluent wastewater, as well as surface water. Codeine, methadone, and its metabolite were determined in all samples of wastewater and the metabolite of cocaine, benzoylecgonine, was found at the highest concentration.     

Imatinib assay by high‐performance liquid chromatography in tandem mass spectrometry with solid‐phase extraction in human plasma

We have developed a method of liquid chromatography in tandem with mass spectrometry to monitor therapeutic levels of imatinib in plasma, a selective inhibitor of protein tyrosine kinase. After solid‐phase extraction of plasma samples, imatinib and its internal standard, imatinib‐D8, were eluted with Zorbax SB‐C₁₈ at 60 °C, under isocratic conditions through a mobile phase consisting of 4 mm ammonium formate, pH: 3.2 (solution A) and acetonitrile solution B. The flow rate was 0.8 mL/min with 55% solution A + 45% solution B. Imatinib was detected and quantified by mass spectrometry with electrospray ionization operating in selected‐reaction monitoring mode. The calibration curve was linear in the range 10–5000 ng/mL, the lower limit of quantitation being 10 ng/mL. The method was validated according to the recommendations of the Food and Drug Administration, including tests of matrix effect (bias < 10%) and recovery efficiency (>80 and <120%). The method is precise (coefficient of variance intra‐day <2% and inter‐day <7%), accurate (95–108%), sensitive and specific. It is a simple method with very fast recording time (1.2 min) that is applicable to clinical practice. This will permit improvement of the pharmacological treatment of patients. Copyright © 2012 John Wiley & Sons, Ltd.     

Metabolomics method based on ultra high performance liquid chromatography with time‐of‐flight mass spectrometry to analyze toxins in fresh and dried toad venom

Drying is a critical step to prolong the storage time in natural medicine processing but it changes the chemical characteristics of the product. In this study, research was performed to characterize the metabolomic changes in toad venom induced by vacuum‐drying at 60°C and air‐drying at room temperature by ultra high performance liquid chromatography coupled with pattern recognition approaches. In total 52 metabolites, down‐regulated or up‐regulated, were identified as potential chemical markers. Compared with fresh toad venom, vacuum‐drying at 60°C succeeded in raising the conjugated‐type bufadienolide content significantly, while the content of free‐type bufadienolides were slightly reduced. On the other hand, toad venom air‐dried at room temperature presented a relatively low amount of bufadienolides compared with fresh venom. For example, the content of several known anti‐tumor components (gamabufotalin, bufotalin, cinobufagin, etc.) were significantly reduced. The 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide bioassay further showed that venom air‐dried at room temperature had weaker anti‐tumor activity on human hepatocellular carcinoma SMMC‐7721 proliferation in vitro than samples vacuum‐dried at 60°C. These results showed that the great metabolomic changes of toad venom occurred during the drying process, suggesting that a proper drying procedure is important for sustaining the chemical quality of natural medicines.     

Improved sample preparation of glyphosate and methylphosphonic acid by EPA method 6800A and time‐of‐flight mass spectrometry using novel solid‐phase extraction

The employment of chemical weapons by rogue states and/or terrorist organizations is an ongoing concern in the United States. The quantitative analysis of nerve agents must be rapid and reliable for use in the private and public sectors. Current methods describe a tedious and time‐consuming derivatization for gas chromatography–mass spectrometry and liquid chromatography in tandem with mass spectrometry. Two solid‐phase extraction (SPE) techniques for the analysis of glyphosate and methylphosphonic acid are described with the utilization of isotopically enriched analytes for quantitation via atmospheric pressure chemical ionization–quadrupole time‐of‐flight mass spectrometry (APCI‐Q‐TOF‐MS) that does not require derivatization. Solid‐phase extraction‐isotope dilution mass spectrometry (SPE‐IDMS) involves pre‐equilibration of a naturally occurring sample with an isotopically enriched standard. The second extraction method, i‐Spike, involves loading an isotopically enriched standard onto the SPE column before the naturally occurring sample. The sample and the spike are then co‐eluted from the column enabling precise and accurate quantitation via IDMS. The SPE methods in conjunction with IDMS eliminate concerns of incomplete elution, matrix and sorbent effects, and MS drift. For accurate quantitation with IDMS, the isotopic contribution of all atoms in the target molecule must be statistically taken into account. This paper describes two newly developed sample preparation techniques for the analysis of nerve agent surrogates in drinking water as well as statistical probability analysis for proper molecular IDMS. The methods described in this paper demonstrate accurate molecular IDMS using APCI‐Q‐TOF‐MS with limits of quantitation as low as 0.400 mg/kg for glyphosate and 0.031 mg/kg for methylphosphonic acid. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination and qualitative analysis of six types of components in Naoxintong capsule by miniaturized matrix solid‐phase dispersion extraction coupled with ultra high‐performance liquid chromatography with photodiode array detection and quadrupole time‐of‐flight mass spectrometry

A simple and effective sample preparation process based on miniaturized matrix solid‐phase dispersion was developed for simultaneous determination of phenolic acids (gallic acid, chlorogenic acid, ferulic acid, 3,5‐dicaffeoylqunic acid, 1,5‐dicaffeoylqunic acid, rosmarinic acid, lithospermic acid, and salvianolic acid B), flavonoids (kaempferol‐3‐O‐rutinoside, calycosin, and formononetin), lactones (ligustilide and butyllidephthalide), monoterpenoids (paeoniflorin), phenanthraquinones (cryptotanshinone), and furans (5‐hydroxymethylfurfural) in Naoxintong capsule by ultra high‐performance liquid chromatography. The optimized condition was that 25 mg Naoxintong powder was blended homogeneously with 100 mg Florisil PR for 4 min. One milliliter of methanol/water (75:25, v/v) acidified by 0.05% formic acid was selected to elute all components. It was found that the recoveries of the six types of components ranged from 61.36 to 96.94%. The proposed miniaturized matrix solid‐phase dispersion coupled with ultra high‐performance liquid chromatography was successfully applied to simultaneous determination of the six types of components in Naoxintong capsules. The results demonstrated that the proposed miniaturized matrix solid‐phase dispersion coupled with ultra high‐performance liquid chromatography could be used as an environmentally friendly tool for the extraction and determination of multiple bioactive components in natural products.     

Simultaneous determination of seven compounds in Chinese patent medicines Chenxiangqu by matrix solid‐phase dispersion coupled with ultra high performance liquid chromatography and quadrupole time‐of‐flight mass spectrometry

A simple, efficient, and sensitive strategy by coupling matrix solid‐phase dispersion with ultra high performance liquid chromatography quadrupole time‐of‐flight mass spectrometry was proposed to extract and determine three types of components (including seven analytes) in Chinese patent medicines Chenxiangqu. The highly ordered mesoporous material Fe‐SBA‐15 synthesized under weakly acidic conditions was selected as a dispersant in matrix solid phase dispersion extraction for the first time. Several parameters including the mass ratio of sample to dispersant, the type of dispersant, the grinding time, and the elution condition were investigated in this work. Under the optimized conditions, 20 compounds were identified by quadrupole time‐of‐flight mass spectrometry and seven analytes were quantified. The results demonstrated that the developed method has good linearity (r > 0.9995), and the limits of detection of the analytes were as low as 0.55 ng/mL. The recoveries of all seven analytes ranged from 97.6 to 104.6% (relative standard deviation < 3.4%). Finally, the improved method was successfully applied to determination of five batches of Chenxiangqu samples, which provided a robust method in quality control of Chinese patent medicines Chenxiangqu. The developed strategy also shows its great potential in analysis of complex matrix samples.     

Simultaneous determination of six toxic alkaloids in human plasma and urine using capillary zone electrophoresis coupled to time‐of‐flight mass spectrometry

A novel capillary zone electrophoresis separation coupled to electro spray ionization time‐of‐flight mass spectrometry method was developed for the simultaneous analysis of six toxic alkaloids: brucine, strychnine, atropine sulfate, anisodamine hydrobromide, scopolamine hydrobromide and anisodine hydrobromide in human plasma and urine. To obtain optimal sensitivity, a solid‐phase extraction method using Oasis MCX cartridges (1 mL, 30 mg; Waters, USA) for the pretreatment of samples was used. All compounds were separated by capillary zone electrophoresis at 25 kV within 12 min in an uncoated fused‐silica capillary of 75 μm id × 100 cm and were detected by time‐of‐flight mass spectrometry. This method was validated with regard to precision, accuracy, sensitivity, linear range, limit of detection (LOD), and limit of quantification (LOQ). In the plasma and urine samples, the linear calibration curves were obtained over the range of 0.50–100 ng/mL. The LOD and LOQ were 0.2–0.5 ng/mL and 0.5–1.0 ng/mL, respectively. The intra‐ and interday precision was better than 12% and 13%, respectively. Electrophoretic peaks could be identified by mass analysis.     

Characterization of tropane and cinnamamide alkaloids from Scopolia tangutica by high‐performance liquid chromatography with quadrupole time‐of‐flight tandem mass spectrometry

Scopolia tangutica is a traditional Chinese medicine used for antispasmodic, anesthesia, analgesia, and sedation. Its medicinal activity is associated to alkaloid constituents, including tropane and cinnamamide types. Low content of alkaloids in plant makes them difficult to be isolated and identified. The present work developed an effective method to quickly characterize alkaloids from Scopolia tangutica by high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry. Thirteen reference compounds were studied for their fragmentation pathways, including five tropane alkaloids and eight cinnamamide ones. Alkaloid constituent was analyzed by an optimized high‐performance liquid chromatography method and mass spectrometry analysis to achieve systematic characterization of alkaloids from Scopolia tangutica. As a result, 53 compounds were identified, including 21 tropane alkaloids (eight new ones), 18 caffeoyl ones (ten new ones) and 14 dicaffeoyl ones (seven new ones). It was important to provide rich information in phytochemical study and structure‐guided isolation of important compounds from this plant.     

Analysis of nitrogenous organic compounds from mainstream cigarette smoke using low‐temperature solvent extraction followed by comprehensive two‐dimensional gas chromatography with high‐resolution time‐of‐flight mass spectrometry

A method involving comprehensive two‐dimensional gas chromatography coupled to high‐resolution time‐of‐flight mass spectrometry was developed and applied to the analysis of nitrogenous organic compounds present in mainstream cigarette smoke trapped on self‐designed equipment. The samples were prepared using low‐temperature solvent extraction under liquid nitrogen and analyzed by comprehensive two‐dimensional gas chromatography with high‐resolution time‐of‐flight mass spectrometry. Important experimental parameters, such as the type and volume of the extraction solvent and flow rate of smoking, were optimized to improve the analysis parameter. The results indicated that 180 mL of diethyl ether in the low‐temperature solvent extraction apparatus system with a 4 mL/min smoke flow rate were the optimal conditions. Then, 85 nitrogenous organic compounds were identified and quantified using a mass spectral library search, accurate mass ion and N‐rules of a molecular formula for nitrogen compounds. Finally, a comparison of the low temperature solvent extraction method and Cambridge filter pad method indicated that more peaks, a higher peak volume and better repeatability were obtained using the low‐temperature solvent extraction method.     

Solid acids assisted matrix solid‐phase dispersion microextraction of alkaloids by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry

The quantification of three alkaloids is important because quantitative study is a means of assessing the reliability of the experimental method, and three alkaloids of peimine, peiminine, and peimisine are main active ingredients in Chinese Pharmacopoeia 2015. An effective method based on the matrix solid‐phase dispersion microextraction was developed for the extraction of alkaloid compounds in Fritillariae Thunbergii Bulbus. Target analytes were analyzed by capillary electrophoresis coupled with quadrupole time‐of‐flight mass spectrometry. The optimized experimental condition was that 50 mg Fritillariae Thunbergii Bulbus was blended homogeneously with 10 mg citric acid for 5 min. Two hundred microliters of water acidized by 1 mol/L hydrochloric acid (pH = 4.5) was selected to elute tested alkaloids. The results demonstrated that the investigated method had low limits of detection (1.32–1.59 ng/mL), good recoveries (86.63–98.12%), and reproducibility (relative standard deviations of peak areas < 0.87%). The proposed matrix solid‐phase dispersion microextraction coupled with capillary electrophoresis combined with quadrupole time‐of‐flight mass spectrometry was successfully applied for the extraction of alkaloids in plants.     

Metabolic profiling of quercetin in rats using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry

Quercetin, a kind of major flavonoid found in many traditional chinese medicines, is an effective substance for treatments such as lowering blood lipids. However, the studies on quercetin have been mainly focused on its pharmacological effect; the treatment of diseases on a material basis, particularly the metabolites derived from quercetin in vivo , has not been evaluated. In this study, we determined the levels, distributions and types of quercetin's metabolites in plasma, urine, feces and bile of rats after a single oral administration of quercetin at a dose of 80 mg/kg, using ultra‐performance liquid chromatography/quadrupole‐time‐of‐flight mass spectrometry (UPLC‐Q‐TOF/MS). A total of 36 metabolites of quercetin were identified, including 11 metabolites in plasma, 34 metabolites in urine, 12 metabolites in feces and 21 metabolites in bile. The results showed that phase I metabolites were reduction metabolites and phase II metabolites mainly included glucuronidation, sulfation and methylation metabolites. These results provide important information on the metabolism of quercetin, which will be helpful for its further development and utilization.     

Development and validation of a magnetic solid‐phase extraction with high‐performance liquid chromatography method for the simultaneous determination of amphetamine and methadone in urine

The simultaneous determination of amphetamine and methadone was carried out by magnetic graphene oxide nanoparticles, a magnetic solid‐phase extraction adsorbent, as a new sample treatment technique. The main factors (the amounts of sample volume, amount of adsorbent, type and amount of extraction organic solvent, time of extraction and desorption, pH, the ionic strength of extraction medium, and agitation rate) influencing the extraction efficiency were investigated and optimized. Under the optimized conditions, good linearity was observed in the range of 100–1500 ng/mL for amphetamine and 100–1000 ng/mL for methadone. The method was evaluated for determination of AM and methadone in positive urine samples, satisfactory results were obtained, therefore magnetic solid‐phase extraction can be applied as a novel method for the determination of drugs of abuse in forensic laboratories.     

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.     

In vitro metabolism study of Strychnos alkaloids using high‐performance liquid chromatography combined with hybrid ion trap/time‐of‐flight mass spectrometry

In this report, the in vitro metabolism of Strychnos alkaloids was investigated using liquid chromatography/high‐resolution mass spectrometry for the first time. Strychnine and brucine were selected as model compounds to determine the universal biotransformations of the Strychnos alkaloids in rat liver microsomes. The incubation mixtures were separated by a bidentate‐C₁₈ column, and then analyzed by on‐line ion trap/time‐of‐flight mass spectrometry. With the assistance of mass defect filtering technique, full‐scan accurate mass datasets were processed for the discovery of the related metabolites. The structural elucidations of these metabolites were achieved by comparing the changes in accurate molecular masses, calculating chemical component using Formula Predictor software and defining sites of biotransformation based upon accurate MSⁿ spectral information. As a result, 31 metabolites were identified, of which 26 metabolites were reported for the first time. These biotransformations included hydroxylation, N‐oxidation, epoxidation, methylation, dehydrogenation, de‐methoxylation, O‐demethylation, as well as hydrolysis reactions. Copyright © 2013 John Wiley & Sons, Ltd.     

Potential of monitoring isotopologues by quantitative gas chromatography with time‐of‐flight mass spectrometry for metabolomic assay

Because of the extreme complexity of metabolomic samples, the effectiveness of quantitative gas chromatography with time‐of‐flight mass spectrometry depends substantially on the expansion of the linear dynamic range. Facing the existence of numerous saturated detector signals, a data processing method based on monitoring isotopologues has been developed. The monoisotopic ion kept the high mass spectrometry sensitivity, and the less abundant isotopologue ions extended the linear dynamic range. This alternative method was proved to extend the linear dynamic range to five orders of magnitude successfully and overcome the quantitative problems induced by the ion detector saturation. Finally, to validate the applicability, the method was applied to a metabolomic assay of Alzheimer's disease. Comparing with the traditional monoisotopic method, the use of monitoring isotopologues helped us to discover an additional eight metabolites with significant difference and to conduct a more reliable principal component analysis as well. The results demonstrated that monitoring isotopologues in quantitative gas chromatography with time‐of‐flight mass spectrometry could improve the authenticity of metabolomic analysis.     

New potential biomarkers for mesterolone misuse in human urine by liquid chromatography quadrupole time‐of‐flight mass spectrometry

In this paper, mesterolone metabolic profiles were investigated carefully. Mesterolone was administered to one healthy male volunteer. Urinary extracts were analyzed by liquid chromatography quadruple time‐of‐flight mass spectrometry (LC‐QTOFMS) for the first time. Liquid–liquid extraction was applied to processing urine samples, and dilute‐shoot analyses of intact metabolites were also presented. In LC‐QTOFMS analysis, chromatographic peaks for potential metabolites were hunt down by using the theoretical [M–H]^? as target ions in full scan experiment, and their actual deprotonated ions were analyzed in targeted MS/MS mode. Ten metabolites including seven new sulfate and three glucuronide conjugates were found for mesterolone. Because of no useful fragment ion for structural elucidation, gas chromatography–mass spectrometry instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after solvolysis. Thus, their potential structures were proposed particularly by a combined MS approach. All the metabolites were also evaluated in terms of how long they could be detected, and S1 (1α‐methyl‐5α‐androst‐3‐one‐17β‐sulfate) together with S2 (1α‐methyl‐5α‐androst‐17‐one‐3β‐sulfate) was detected up to 9 days after oral administration, which could be the new potential biomarkers for mesterolone misuse. Copyright © 2015 John Wiley & Sons, Ltd.     

Separation and identification of phenolic compounds in Bidens pilosa L. by ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A validated method based on ultra‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry was established to separate and identify phenolic compounds in Bidens pilosa L. Mass spectrometry experiments were performed both in positive and negative ion modes. A total of 35 compounds were detected, and 26 phenolic compounds were unequivocally identified or tentatively assigned based on retention time, maximum UV absorption, molecular formula, and fragments. The ultra high performance liquid chromatography method was validated and showed good linearity (R² ≧ 0.9996) over the test range. The limits of detection and quantification were above 0.072 and 0.162 μg/mL, respectively. The relative standard deviations of intraday and interday precision were below 0.3 and 1.6%, respectively.     

High‐throughput 96‐well solid‐phase extraction for preparation of tetracycline followed by liquid chromatography and mass spectrometry analysis

Tetracyclines abuse has frequently occurred in aquaculture against bacteria, rickettsiae, spirochetes, and mycoplasmas. In this study, a high‐throughput sample preparation method was developed using 96‐well plate solid‐phase extraction (p‐SPE) and the extract was analyzed by ultra‐performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS). The experimental conditions were optimized such that the pH is 4, the eluting solvent is methanol (2 mL), and the sorbent is hydrophilic‐lipophilic balance (HLB) microsphere. The whole protocol was validated, and it showed that the tetracyclines were linear with correlation coefficients ≥ 0.9990, precision and accuracy (RSD%) in 3.9–6.1%, and mean recoveries of 88.6–103.6%. To exhibit the potential of 96‐well p‐SPE as a routine tool for inspection and quarantine, fresh aquatic samples were tested, and among which positive samples were observed. This method was demonstrated to be promising for the purification and enrichment of tetracyclines with reduced time and labor, and indeed practically and particularly suitable for widespread tetracyclines analysis.