Information-dependent Acquisition Research Articles

In Vitro Metabolite Profiling of ADB-FUBINACA, A New Synthetic Cannabinoid.

Metabolite profiling of novel psychoactive substances (NPS) is critical for documenting drug consumption. N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (ADB-FUBINACA) is an emerging synthetic cannabinoid whose toxicological and metabolic data are currently unavailable. We aimed to determine optimal markers for identifying ADB-FUBINACA intake. Metabolic stability was evaluated with human liver microsome incubations. Metabolites were identified after 1 and 3 h incubation with pooled human hepatocytes, liquid chromatography- high resolution mass spectrometry in positive-ion mode (5600+ TripleTOF®, Sciex) and several data mining approaches (MetabolitePilot™, Sciex). Metabolite separation was achieved on an Ultra Biphenyl column (Restek®); full-scan TOF-MS and information-dependent acquisition MS/MS data were acquired. ADB-FUBINACA microsomal half-life was 39.7 min, with a predicted hepatic clearance of 9.0 mL/min/kg and a 0.5 extraction ratio (intermediate-clearance drug). Twenty-three metabolites were identified. Major metabolic pathways were alkyl and indazole hydroxylation, terminal amide hydrolysis, subsequent glucuronide conjugations, and dehydrogenation. We recommend ADB-FUBINACA hydroxyalkyl, hydroxydehydroalkyl and hydroxylindazole metabolites as ADB-FUBINACA intake markers. N-dealkylated metabolites are not specific ADB-FUBINACA metabolites and should not be used as definitive markers of consumption. This is the first ADB-FUBINACA in vitro metabolism study; in vivo experiments enabling pharmacokinetic and pharmacodynamics studies or urine from authentic clinical/forensic cases are needed to confirm our results.

Pharmacokinetics of Cajaninstilbene Acid and Its Main Glucuronide Metabolite in Rats.

As a major active stilbene from the leaves of pigeon pea (Cajanus cajan), cajaninstilbene acid (CSA) exerts various pharmacological activities. The present study aimed to investigate the pharmacokinetics of CSA and one of its main metabolites (M1) to explore their fate in the body and provide a pharmacokinetic foundation for their in vivo biological activities and functional food or complementary medicine application. M1 was characterized as CSA-3-O-glucuronide using the multiple reaction monitoring-information-dependent acquisition-enhanced product ion technique. After oral and intravenous administration, plasma, urine, and bile were collected and analyzed to estimate pharmacokinetic properties of CSA and M1 and to explore the main excretion route. The oral bioavailability of CSA was estimated to be 44.36%. This study first reported that CSA is mainly metabolized to CSA-3-O-glucuronide via the first-pass effect to limit its oral bioavailability and excreted predominantly through the biliary route, while the enterohepatic circulation, extravascular distribution, and renal reabsorption characteristics of CSA might delay its elimination.

Quantification of 11-nor-9-carboxy-THC and panel of 22 drugs in hair using a hybrid triple quadrupole linear ion trap mass spectrometer (QTRAP)

The goal of this study was to develop a mass spectrometric method that reproducibly detects and quantifies 11-nor-9-Carboxy-THC levels in hair below 0.2 pg/mg. Using the same QTRAP system, a method was evaluated that allows the identification with simultaneous quantification and confirmation through MS/MS library matching and ion ratio values, for a panel of commonly analyzed forensic drugs in hair. Sample preparation of 11-nor-9-Carboxy-THC consisted of a methanol/water wash followed by a NaOH digest at 75 °C for one hour and then hexane/ethyl acetate liquid extraction. For the other compounds an overnight acetonitrile/formic acid digest was used. HPLC separation used a reverse-phase column at 30 °C, using 8.0 minute gradient. A SCIEX 6500 + QTRAP system in MRM3 mode was used for 11-nor-9-Carboxy-THC detection and MRM (two transitions per analyte) with Information Dependent Acquisition (IDA)-triggering enhanced product ion (MRM-IDA-EPI) scans for analysis of all other analytes. Detection of 11-nor-9-Carboxy-THC in hair by MRM is hampered by the complexity of the matrix and presence of compounds of similar structure and mass. This often leads to high background and interfering peaks in the MRM trace that does not allow the low detection requirements. In order to eliminate these interferences and background to improve the LOQ with good reproducibility, MRM3 fragmentation has the required selectivity to allow for the low level quantification. We will show that at the 0.2 pg/mg cutoff level in MRM mode the 11-nor-9-Carboxy-THC is hardly detectable in the high background and multiple interfering peaks but easily detectable using the MRM3 approach. We were able to quantifiy 11-nor-9-Carboxy-THC levels in hair down to 0.04 pg/mg with 99% accuracy and less than 11% CV. A scheduled MRM-IDA-EPI experiment was developed to detect and confirm the presence of analytes from a targeted 22 compound panel in hair. The experiment allowed the simultaneous confirmation via MS/MS library matching (scores > 75% for all compounds) and ion ratios (<20% CV for all analytes) as well as successful quantification of each compound in the panel with high precision. This was achieved by scheduling the MRM transitions and maximizing the time spent acquiring quality full scan MS/MS data to use in library searching. Linearity was from 5 pg/mg to 2.5 ng/mg for all compounds. We developed a MRM3 method that reproducibly detects and quantifies 11-nor-9-Carboxy-THC levels in hair down to 0.04 pg/mg. We also developed a QTRAP method allowing simultaneous identification quantification and confirmation for a panel of 22 forensic drug compounds. For Research Use Only. Not for use in diagnostic procedures.

Ultra-fast forensic toxicological screening and quantitation under 3 minutes with a QTOF HPLC-MS/MS system

Objectives HPLC-MS/MS technology combines separation (HPLC) and detection (MS, and MS/MS) and is widely used for screening applications. Mostly, the duration of HPLC runtime in these studies vary from 5 to 20 min. For high-throughput laboratories, a fast screening method is desired. There are mainly two approaches to acquire fragmentation information for screening purpose of compounds by HPLC-MS/MS: Information-Dependent-Acquisition (IDA) or Data-Dependent-Acquisition (DDA) and MS/MS All . For IDA-MS/MS, a survey scan is performed to collect the information on the precursor ions, followed by multiple dependent MS/MS scans for a number of most abundant precursor/candidate ions. MS/MS All with SWATH ® acquisition is a novel MS/MS All technique. In every data cycle, the instrument acquires TOF-MS information first, and then it sequentially acquires MSMS information of all precursor ions across a specified mass range in pre-divided mass windows. Both of these two data acquisition approaches are compatible with retrospective data interrogation. In this study, we aimed to develop an ultra-fast screening method in a forensic toxicological setting using the SCIEX X500R QTOF system with SCIEX OS software 1.0. We also aim to compare IDA-MS/MS and MS/MS All with SWATH ® acquisition for this fast screening method. Methods Blank human urine samples were spiked with multiple drugs commonly found in forensics setting at different concentration levels. Typically, samples were diluted 10-fold in 10% methanol and centrifuged. The clear supernatants were transferred to auto-sampler vials and 10 μL samples were injected. HPLC separation was on a 20 × 2 mm cartridge-type column with cartridge holder. Mobile phase A was 10 mm ammonium formatted in water and mobile phase B was 0.1% formic acid in methanol. HPLC runtime was 2.5 min Data was acquired, processed and reported in SCIEX OS software 1.0. Results Good separation for all the analytes tested and good retention for polar compounds. Sample set 1 was blank urine samples spiked with more than 85 compounds at different levels (CO1 means cutoff number for Sample set 1). Despite a very short HPLC runtime (less than 3 min), the method showed excellent sensitivity. The calibration curve for one of the analytes (JWH 210 5-OH metabolite) is showed. Sample set 2 were also urine samples with both calibrators (spiked with a different group of 84 compounds, CO 2 means cutoff number for sample set 2) and unknown samples. It showed true positive rate in the calibrators for set 2. No false positive was identified. It showed the true positive rate with different HPLC methods and MS/MS acquisition methods. Conclusion In this study, we have developed a super-fast screening/quantitation method in forensic setting under 3 minutes using the SCIEX X500R QTOF system. Two non-targeted data acquisition methods: IDA-MS/MS and MS/MS All with SWATH ® acquisition were both tested and compared. Depending on the specific requirement of the screening method, both IDA-MS/MS and SWATH® acquisition have their advantage and disadvantage in certain areas and the user should be flexible to adopt either approach with appropriate method settings. For Research Use Only. Not for use in diagnostic procedures.

In vitro metabolism of new synthetic cannabinoid SDB-006 in human hepatocytes by high-resolution mass spectrometry

The drug abuse epidemic within the United States remains one of the nation’s most serious social challenges, especially among adolescents and young adults. Novel psychoactive substances continuously emerge into the illicit drugs-of-abuse market to evade legislation. In 2013, SDB-006 was detected as a novel synthetic cannabinoid (SC) with high binding affinity to CB1 (EC50 = 19 nM) and CB2 (EC50 = 134 nM). Unfortunately, no human metabolism data for SDB-006 are currently available, making it challenging to confirm intake, since all previously investigated SCs were extensively metabolized. The present study aims to recommend appropriate marker metabolites for documenting SDB-006 consumption by investigating its metabolism in human hepatocytes. For metabolite profiling, 10 µM of SDB-006 was incubated in human hepatocytes for 3 h. Metabolite identification in hepatocyte samples was accomplished with high-resolution mass spectrometry via information-dependent data acquisition. Results revealed that SDB-006 was highly metabolized in human hepatocytes. A total of 20 metabolites were characterized, generated mainly from hydroxylation and glucuronidation. Hydroxylation occurred primarily on several positions of the pentyl chain. N-Dealkylation was the other major pathway, including depentylation and debenzylation. Based on our data, we propose 4′-keto-SDB-006 (M19) and pentyl-OH-SDB-006 (M15) as optimal marker metabolites for documenting SDB-006 intake.

Identification of Oxidative Degradation Products of Lansoprazole by Using High Resolution Mass Spectrometry Spectral Data

The study focused on the application of high-resolution mass spectrometry for the identification of impurities in pharmaceutical small molecules. A high-performance liquid chromatography (HPLC) coupled high resolution mass spectrometer (HRMS) was used for identification of oxidative degradation impurities (DIs) of lansoprazole. The utilization of HRMS facilitates to determine the accurate mass of impurities and their fragment/product ions. A fast mass spectrometer (MS) compatible reverse phase chromatography method was used to investigate the oxidative stressed impurities. HPLC column; C18 (50 × 4.6 mm, 3.5 μm) was used with gradient elution. Spectral data acquired using information dependent acquisition (IDA) with real time dynamic background subtraction algorithm (DBS). Three oxidative impurities: DI-I (m/z 386.0781), DI-II (m/z 402.0734) and DI-III (m/z 386.0785), was observed during this study; interpretation of high resolution spectral data of all three impurities was carried out; elemental composition and molecular structure was proposed for major fragments. In this study mass error was found ≤7.7 parts per million (ppm).

Rapid and highly sensitive analysis of benzodiazepines and tandospirone in human plasma by automated on-line column-switching UFLC-MS/MS.

A high-throughput method was developed for the detection of 31 benzodiazepine drugs and tandospirone in human plasma by on-line column-switching ultra-fast liquid chromatography-tandem mass spectrometry. Plasma samples (100μl) spiked with the 32 drugs and oxazepam-d5 (internal standard) were diluted with 300μl of 13.3mM ammonium acetate/acetonitrile (33:67, v/v). After centrifugation and filtration, the clear supernatant was injected directly onto the extraction column (Oasis HLB cartridge column). The following procedure was fully automated. The analytes retained on the extraction column were eluted by backflushing of the extraction column and introduced into an analytical column (SUMIPAX ODS D-Swifter column, 30mm×3.0mm i.d.; particle size 2μm) by column switching. Quantification was performed by multiple reaction monitoring with positive-ion electrospray ionization. Distinct peaks appeared for each drug and the internal standard on each channel within 7min, including the extraction time. All drugs spiked into plasma showed recoveries of 83-95%. The regression equations for the 32 drugs showed excellent linearities in the range of 50-2000pg/ml of plasma and the limits of detection ranged from 20 to 50pg/ml. The lower and upper limits of quantitation were 50-100ng/ml and 2000pg/ml, respectively. Intra- and interday coefficients of variation for none of the drugs were greater than 13.6%. The accuracies of quantitation were 87-112%. The multiple reaction monitoring information-dependent acquisition of enhanced product ions method enabled the quantification and confirmation of diazepam, triazolam, and lorazepam obtained from actual plasma.

Wide-scope screening of pesticides in fruits and vegetables using information-dependent acquisition employing UHPLC-QTOF-MS and automated MS/MS library searching

This paper presents an application of ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) for simultaneous screening and identification of 427 pesticides in fresh fruit and vegetable samples. Both full MS scan mode for quantification, and an artificial-intelligence-based product ion scan mode information-dependent acquisition (IDA) providing automatic MS to MS/MS switching of product ion spectra for identification, were conducted by one injection. A home-in collision-induced-dissociation all product ions accurate mass spectra library containing more than 1700 spectra was developed prior to actual application. Both qualitative and quantitative validations of the method were carried out. The result showed that 97.4% of the pesticides had the screening detection limit (SDL) less than 50μg kg-1 and more than 86.7% could be confirmed by accurate MS/MS spectra embodied in the home-made library. Meanwhile, calibration curves covering two orders of magnitude were performed, and they were linear over the concentration range studied for the selected matrices (from 5 to 500μg kg-1 for most of the pesticides). Recoveries between 80 and 110% in four matrices (apple, orange, tomato, and spinach) at two spiked levels, 10 and 100μg kg-1, was 88.7 or 86.8%. Furthermore, the overall relative standard deviation (RSD, n = 12) for 94.3% of the pesticides in 10μg kg-1 and 98.1% of the pesticides in 100μg kg-1 spiked levels was less than 20%. In order to validate the suitability for routine analysis, the method was applied to 448 fruit and vegetable samples purchased in different local markets. The results show 83.3% of the analyzed samples have positive findings (higher than the limits of identification and quantification), and 412 commodity-pesticide combinations are identified in our scope. The approach proved to be a cost-effective, time-saving and powerful strategy for routine large-scope screening of pesticides.

Identification of in vitro and in vivo metabolites of alantolactone by UPLC-TOF-MS/MS.

Alantolactone (AL), an active sesquiterpene originating from Inula helenium, is a potential anticancer and anti-inflammatory agent. However so far, studies on AL metabolism have not been reported. In the present study, we have investigated for the first time the in vivo and in vitro metabolites of AL using ultra performance liquid chromatography combined with time of flight mass spectrometry (UPLC-TOF-MS/MS). A unique on-line information-dependent acquisition (IDA) method multiple mass defect filter (MMDF) combined with dynamic background subtraction (DBS) was applied to trace all of the probable metabolites of AL. Five MMDF templates were set according to the core structure of AL and the general metabolite biotransformation patterns, and other five sulfur-containing dimer filter templates were first established on the basis of structural elucidation of AL metabolites obtained from rat intestinal bacteria biotransformation. As a result, 44 metabolites were characterized: 41 metabolites from rat urine, bile and feces after oral administration of AL, and 13 metabolites from AL biotransformation by rat intestinal bacteria. Particularly, 26 metabolites were identified as novel sulfur-containing products. The results indicated that addition of double bond at Δ((11,13)) and oxidization were the main metabolic reactions of AL. A new metabolism pathway to produce addition products of H2S to AL and further generate a series of sulfur-containing dimers of AL was revealed. This study significantly enriched our knowledge about AL metabolism, which will lead to a better understanding of the safety and efficacy of AL. At the same time, the established methodology can be widely applied for the structural determination of the metabolites of other sesquiterpene containing α-methylene-γ-lactone moiety.

Simultaneous identification and quantification of tetrodotoxin in fresh pufferfish and pufferfish-based products using immunoaffinity columns and liquid chromatography/quadrupole-linear ion trap mass spectrometry

In this study, we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin (TTX) in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry (LC-QqLIT-MS). TTX was extracted by 1% acetic acid-methanol, and most of the lipids were then removed by freezing lipid precipitation, followed by purification and concentration using immunoaffinity columns (IACs). Matrix effects were substantially reduced due to the high specificity of the IACs, and thus, background interference was avoided. Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase. The method was evaluated by fortifying samples at 1, 10, and 100 ng/g, respectively, and the recoveries ranged from 75.8%–107%, with a relative standard deviation of less than 15%. The TTX calibration curves were linear over the range of 1–1 000 μg/L, with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g. Using this method, samples can be further analyzed using an information-dependent acquisition (IDA) experiment, in the positive mode, from a single liquid chromatography-tandem mass spectrometry injection, which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion (EPI) library. The scheduled multiple reaction monitoring method enabled TTX to be screened for, and TTX was positively identified using the IDA and EPI spectra. This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products. The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility, and is suitable for the analysis of complex matrix pufferfish samples.

Qualitative screening of absorbed indoloquinazoline alkaloids and their metabolites in rat plasma after the oral administration of Wu-Zhu-Yu decoction by high-resolution mass spectrometry with multiple data mining algorithms.

A rapid and sensitive liquid chromatography with high-resolution mass spectrometry method with multiple data processing algorithms was developed and applied for the metabolite profiling of evodiamine and its analogous alkaloids in rat plasma after the administration of Wu-Zhu-Yu decoction. All samples were purified using hydrophilic-lipophilic balanced solid-phase extraction cartridges and analyzed by a Sciex TripleTOF 5600(+) mass spectrometer with a 35 min liquid chromatography gradient elution. High-resolution full-scan mass spectrometry and information-dependent acquisition tandem mass spectrometry data were analyzed using multiple data processing approaches. The results indicated that the detected eight prototype alkaloids could be metabolized to 58 metabolites through both phase I and phase II reactions. Oxidation was demonstrated to be the principle metabolic pathway of the parent compounds. The study contributes to the understanding of the absorption and metabolism of the alkaloids in Wu-Zhu-Yu decoction and provides a detailed analysis of scientific data.

Analysis of psychoactive substances in water by information dependent acquisition on a hybrid quadrupole time-of-flight mass spectrometer

Emerging drugs of abuse, belonging to many different chemical classes, are attracting users with promises of “legal” highs and easy access via internet. Prevalence of their consumption and abuse through wastewater-based epidemiology can only be realized if a suitable analytical screening procedure exists to detect and quantify them in water. Solid-phase extraction and ultra-high performance liquid chromatography quadrupole time-of-flight-mass spectrometry (UHPLC–QqTOF–MS/MS) was applied for rapid suspect screening as well as for the quantitative determination of 42 illicit drugs and metabolites in water. Using this platform, we were able to identify amphetamines, tryptamines, piperazines, pyrrolidinophenones, arylcyclohexylamines, cocainics, opioids and cannabinoids. Additionally, paracetamol, carbamazepine, ibersartan, valsartan, sulfamethoxazole, terbumeton, diuron, etc. (including degradation products as 3-hydroxy carbamazepine or deethylterbuthylazine) were detected. This method encompasses easy sample preparation and rapid identification of psychoactive drugs against a database that cover more than 2000 compounds that ionized in positive mode, and possibility to identify metabolites and degradation products as well as unknown compounds. The method for river water, influent and effluents samples was fully validated for the target psychoactive substances including assessment of matrix effects (-88–67.8%), recovery (42–115%), precision (<19%) and limits of quantification (1–100ngL−1). Method efficiency was thoroughly investigated for a wide range of waste and surface waters. Robust and repeatable functioning of this platform in the screening, identification and quantification of traditional and new psychoactive drugs biomarkers and other water contaminants is demonstrated.

The development of a quantitative and qualitative method based on UHPLC-QTOF MS/MS for evaluation paclitaxel–tetrandrine interaction and its application to a pharmacokinetic study

Paclitaxel is a broad-spectrum anti-cancer drug by targeting microtubulin. However, multidrug resistant (MDR) makes its clinical application more difficult and results in failure of chemotherapy. Tetrandrine as a potential multidrug resistant modulator could be combined with other anti-cancer drugs. In this study, ultra-performance liquid chromatography (UHPLC) combined with quadrupole time-of-flight mass spectrometry (QTOF) was applied to simultaneously qualitative and quantitative analysis of paclitaxel for the pharmacokinetic studies while combined with tetrandrine. This method was developed based on non-target screening mode IDA (Information Dependent Acquisition). As a result, the validated range was 0.25–64ng/ml (30µl plasma) for paclitaxel. Totally 33 metabolites of paclitaxel and tetrandine were identified in vivo and in vitro. The main metabolites of PTX were dose-dependent decreased with different amounts of tetrandine co-administration no matter in vivo and in vitro, the exposure of PTX increased in pharmacokinetic study. The verified method is sensitive accurate and effective for the simultaneous determination of paclitaxel and its metabolites in blood, urine and live microsome incubation samples and it was successfully applied to evaluate the pharmacokinetics and drug–drug interaction between paclitaxel and tetrandine. Furthermore, a biosensor technology, surface plasmon resonance (SPR) analysis was applied to preliminary evaluate the competitive protein binding of multiple components. The SPR analysis indicated that the affinity between 6-hydroxy-paclitaxel and micotubulin is similar to that between paclitaxel and micotubulin, and tetrandrine also does not form a competitive combination with paclitaxel. For human, 6-hydroxy-paclitaxel is the one of main metabolites of paclitaxel, so the results suggested that tetrandine has an influence on the metabolite of paclitaxel, but tetrandine and the main metabolites of PTX probably do not affect PTX's biological targeting, the effect of its pharmacological action needs to be further studied.

In vitro and in vivo human metabolism of a new synthetic cannabinoid NM-2201 (CBL-2201).

In 2014, NM-2201 (CBL-2201), a novel synthetic cannabinoid (SC), was detected by Russian and United States laboratories. It was already added to the scheduled drugs list in Japan, Sweden and Germany. Unfortunately, no human metabolism data are currently available, making it challenging to confirm its intake because all previous investigated SCs were extensively metabolized. The present study aims to recommend appropriate marker metabolites by investigating NM-2201 metabolism in human hepatocytes and confirm the results in authentic human urine specimens. For the metabolic stability assay, 1 μM NM-2201 was incubated in human liver microsomes (HLMs) for up to 1 h; for metabolite profiling, 10 μM of NM-2201 was incubated in human hepatocytes for 3 h. Two authentic urine specimens from NM-2201 positive cases were analyzed after β-glucuronidase hydrolysis. Metabolite identification in hepatocyte samples and urine specimens was achieved with high-resolution mass spectrometry via information-dependent acquisition. NM-2201 was quickly metabolized in HLMs with an 8.0 min half-life. In human hepatocyte incubation samples, a total of thirteen NM-2201 metabolites were identified, generated mainly from ester hydrolysis and further hydroxylation, oxidative defluorination and subsequent glucuronidation. M13 (5-fluoro PB-22 3-carboxyindole) was the major metabolite. In the urine specimens, the parent drug NM-2201 was not detected; M13 was the predominant metabolite after β-glucuronidase hydrolysis. Therefore, based on our study, we recommend the M13 as a suitable urinary marker metabolite for confirming NM-2201 and/or 5F-PB-22 intake.

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries.

Rotenone Analysis by Liquid Chromatography–Tandem Mass Spectrometry with Information-Dependent Acquisition in a Fatal Case of Rotenone Poisoning with a Commercial Organic Insecticide Being Sold in Korea

Rotenone is a neurotoxin derived from Derris roots or yam bean of genus Derris or Lonchocarpus It is known to cause Parkinson-like symptoms and is a potent electron transport inhibitor. Rotenone was detected in postmortem specimens in a fatal case of rotenone poisoning with an organic pesticide by liquid chromatography-tandem mass spectrometry with an information-dependent acquisition and MS-MS library search. The forensic specimens were prepared by solid-phase extraction with a Bond Elut(®) Certify cartridge. The mobile phase comprised 5 mM ammonium formate in 10% methanol and 5 mM ammonium formate in 90% methanol. The assay was linear over the range from 0.01 to 1.0 mg/L (r(2) = 0.995). The limit of detection and quantitation in the blood were 0.001 mg/L (signal-to-noise, S/N = 3) and 0.003 mg/L (S/N = 10), respectively. The intraday accuracy and precision for rotenone that were determined by five replicates at 0.02, 0.10 and 1.0 mg/L in blood were <15.0% of bias and <9.0% of CV, respectively. The interday accuracy and precision for rotenone that were determined by seven replicates at 0.02, 0.10 and 1.0 mg/L in blood were <18.0% of bias and <17.0% of CV, respectively. Relative recovery with 0.02, 0.1 and 1.0 mg/L in blood was 104.2, 103.3 and 81.6% (n = 6), respectively. The described method was applied for the determination of rotenone in a fatal case of intoxication of a 33-year-old man who was found dead on a bed in a temporary house. In this case study, the concentrations of rotenone in heart blood (HB), peripheral blood (PB), gastric contents and vitreous humor were 0.77 mg/L, 0.02 mg/L, 126.4 mg/kg and 0.003 mg/L, respectively. The rotenone concentration ratio of the HB/PB was 38.8 and that of gastric contents/PB was 6412.3, suggesting a massive ingestion of rotenone with postmortem redistribution. This study is the report of rotenone detection in a fatal case with the ingestion of the organic insecticide containing rotenone.

Efficient forensic toxicological screening and quantitation workflow using QTOF LC–MS/MS system

Quadrupole time-of-flight (QTOF) mass spectrometry is becoming the desired technology for sensitive and selective screening workflows in a forensic toxicological setting. The technology overcomes many challenges faced when using traditional techniques and more significantly captures all information about the sample in one injection to allow for retrospectively mining the data. Here we describe a new benchtop QTOF system with revolutionary N-optic designed flight path and new, intuitive software for easy adoption of accurate mass technology to forensic testing. Blank human urine was spiked with more than 50 common drugs found in forensics setting at different concentration levels. HPLC separation was performed using the SCIEX ExionLC AC system on a reverse-phase column. Data was collected on a SCIEX X500R QTOF mass spectrometer with SCIEX OS software: TOF – MS survey scan with Information Dependent Acquisition (IDA) – triggering of up to 16 product ion scans; or SWATH® acquisition. For SWATH® acquisition, the precursor isolation window width was varied for each MS/MS experiment. Data processing was also performed in SCIEX OS software with simultaneous identification and quantification being accomplished in single software. Screening: multiple confidence criteria including mass accuracy, retention time, isotope, and most importantly, library matching were used. The availability of MS/MS information for identification was required in this study through library searching the acquired spectra against a SCIEX high resolution forensic library containing MS/MS spectra of 1700 compounds. Excellent true positive rates were accomplished for both acquisition modes (89% with IDA and 98% with SWATH® acquisition). This result is consistent with the expectation that the IDA-MS/MS data acquisition has the risk of occasionally missing acquiring MS/MS. The acquisition of MS/MS data in the screening approach allowed the confident identification of structural isomers that have similar retention times through unique fragment ions and their ratios. Simultaneous Quantification Linearity on the new QTOF system for the majority of compounds tested was four orders linear dynamic range covering 0.1 to 1000 ng/mL. SWATH® acquisition ensures that all data, including MS/MS, is acquired at all times. Due to the fast scanning speed (up to 100 Hz at 35 K resolution) in MS/MS mode, a fast cycletime (0.5 sec) was used for the 6.5-min LC method that allowed for one TOF-MS scan and 16 MS/MS scans that covered 120 to 500 m/z range. A sensitive and selective workflow was developed for toxicological compound screening in a forensic setting using the new X500R QTOF system. The newly designed software that accompanies the new benchtop QTOF instrument allows for simultaneous compound identification and quantification in one step all in one place. SCIEX OS software allows the easy adoption of accurate mass technology for routine forensic workflows.

Liquid chromatography/mass spectrometry identification of intermediates and vulcanization products by using squalene as vulcanization model compound.

Sulfur-vulcanized rubber is a three-dimensional polymer network, insoluble in all organic solvents. For this reason, vulcanization products are difficult to study and identify by conventional analytical techniques. To simplify this task, low molecular weight olefins have been used as model compounds (MCs) in place of rubber in vulcanization experiments. In this work, the vulcanization process was investigated using squalene (SQ) as MC. By-products, intermediates and products were separated by semipreparative reversed-phase liquid chromatography (RPLC) with UV detection. Each fraction was collected, concentrated and characterized by flow injection analysis (FIA) and non-aqueous reversed-phase (NARP) LC coupled to positive atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Under the latter conditions, an Information-Dependent Acquisition (IDA) was performed on a linear ion trap mass spectrometer to obtain structural information. Several vulcanized compounds containing up to three SQ molecules, cross-linked with chains involving up to 14 sulfur atoms overall, have been identified along with some of their oxidized products (epoxides and hydroperoxides). The FIA-MS spectra showed peak clusters, each of which included two-three subclusters; the interpretation was complicated by the occurrence of more ion species per product, by the unsaturation grade and by the characteristic isotopic distribution of sulfur. The enhanced product ion scan (EPI) spectra, acquired during the IDA experiments, supported the FIA-MS identification allowing one to count the number of sulfur atoms. The sensitivity of the developed analytical strategy was due to the enrichment factor achieved via semipreparative chromatography and the very good response of the APCI detection. Pattern fragmentation and chromatographic behavior simplified the identification of the cured compounds and their oxidized products, whose occurrence was related to the grade of oxidation of SQ used as reagent. Copyright © 2016 John Wiley & Sons, Ltd.

UFLC-MS/MS analysis of four tanshinone components in Salvia miltiorrhizae after ultrasound-assisted extraction.

A sensitive and rapid ultra-fast liquid chromatography with tandem mass spectrometry (UFLC-MS/MS) method was developed for simultaneous qualitative and quantitative of four characteristic tanshione components including tanshinone IIA, cryptotanshinone, tanshinone I and dihydrotanshinone I in Salvia miltiorrhizae after ultrasound-assisted extraction. By using a C18 column, the four analytes were separated by gradient elution with acetonitrile and water both containing 0.1% formic acid at the flow rate of 0.3mL/min. Multiple-reaction monitoring (MRM) was used for quantification, and an information-dependent acquisition (IDA) method was used to trigger enhanced product ion scans (EPI) for supplementary characteristic identification for qualitative research. Calibration curves showed good linearities with correlation coefficients (r) higher than 0.9990. The method showed high sensitivity with limits of detection (LODs) and quantification (LOQs) less than 0.0002ng/mL and 0.0008ng/mL, respectively, as well as good precision and reproducibility. Mean recoveries for four analytes ranged from 92.5% to 106.2% with relative standard deviations (RSDs) lower than 14.59%. Real application of the developed method in 32 batches of S. miltiorrhizae samples demonstrated that the total contents of four analytes in all samples were in the range of 2.258-52.342mg/g. Ultrasound-assisted extraction technique took a small amount of sample and low time but giving high extraction efficiency. Combining with UFLC-MS/MS method in MRM-IDA-EPI mode, more components in other complicated matrices can be simultaneously analyzed for qualitation and quantitation in one run.

Investigation of 6-O-methyl-scutellarein metabolites in rats by ultra-flow liquid chromatography/quadrupole-time-of-flight mass spectrometry

Context Scutellarin (1) has been widely used in China to treat acute cerebral infarction and paralysis induced by cerebrovascular diseases. However, scutellarin (1) has unstable metabolic characteristics.Objective The metabolic profile of 6-O-scutellarein was studied to determine its metabolic stability in vivo.Materials and methods In this study, a method of UFLC/Q-TOF MS was used to study the 6-O-methyl-scutellarein metabolites in rat plasma, urine, bile and faeces after oral administration of 6-O-methyl-scutellarein (3). One hour after oral administration of 6-O-methyl-scutellarein (3) (34 mg/kg), approximately 1 mL blood samples were collected in EP tubes from all groups. Bile, urine and faeces samples were collected from eight SD rats during 0–24 h after oral administration. The mass defect filtering, dynamic background subtraction and information dependent acquisition techniques were also used to identify the 6-O-methyl-scutellarein metabolites.Results The parent compound 6-O-methyl-scutellarein (3) was found in rat urine, plasma, bile and faeces. The glucuronide conjugate of 6-O-methyl-scutellarein (M1, M2), diglucuronide conjugate of 6-O-methyl-scutellarein (M3), sulphate conjugate of 6-O-methyl-scutellarein (M4), glucuronide and sulphate conjugate of 6-O-methyl-scutellarein (M5), methylated conjugate of 6-O-methyl-scutellarein (M6) were detected in rat urine. M1, M2 and M3 were detected in rat bile. M1 was found in rat plasma and M7 was detected in faeces.Discussion and conclusion Because the parent compound 6-O-methyl-scutellarein (3) was found in rat urine, plasma, bile and faeces, we speculate that 6-O-methyl-scutellarein (3) had good metabolic stability in vivo. This warrants further study to develop it as a promising candidate for the treatment of ischemic cerebrovascular disease.