Enhanced Product Ion Scan Research Articles

Analysis of carbazole alkaloids in Murraya koenigii by means of high performance liquid chromatography coupled to Tandem mass spectrometry with a predictive multi experiment approach

Murraya koenigii (M. koenigii) is recognized as one of the most significant Indian medicinal plants, due to its therapeutic properties. In fact, various biological activities, including anti-inflammatory and antifungal, are attributed to carbazole alkaloids (CAs) and their metabolites, but it is still difficult to achieve a full classification of these compounds because of their heterogeneous structures. For this reason, the development of new strategies for their identification is necessary. In this work, a reliable method, including the simultaneous quantification of three main CAs of Murraya (Mahanimbine, Koenimbine and Koenigicine) and a putative identification for other compounds belonging to the huge family of CAs, was developed by means of HPLC-MS/MS. Also, an efficient extraction procedure followed by a suitable clean-up step was presented, in order to obtain reliable recoveries (resulted from 60 to 85% for all the analytes). The analyses were performed by using predictive multi experiment approach based on information-dependent acquisition (IDA), coupling multiple reaction monitoring (MRM) and precursor ion (PI) as survey scans, and enhanced product ion scan (EPI) as dependent scan. Competitive Fragmentation Modeling-ID (CFM-ID) was used to predict MS/MS spectra from the chemical structures of the compounds in order to create a suitable MRM inclusion list. The obtained results showed that this method can simultaneously provide quantitative information for the target analytes (Mahanimbine (7.22–5.62 mg/kg), Koenimbine (1.26–1.62 mg/kg) and Koenigicine (0.44–1.77 mg/kg)) and a putative identification for several compounds belonging to different classes of CAs which are not included in the target list, thanks to PI survey scan.

Determination of Mycotoxins in Dried Fruits Using LC-MS/MS-A Sample Homogeneity, Troubleshooting and Confirmation of Identity Study.

To monitor co-exposure to toxic mycotoxins in dried fruits, it is advantageous to simultaneously determine multiple mycotoxins using a single extraction and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. In this study, we applied a stable isotope dilution and LC-MS/MS method to multi-mycotoxin analysis in dried fruits, selecting raisins, plums, figs, and cranberries for matrix extension. Samples were prepared using cryogenic grinding, followed by the fortification of carbon-13 (13C) uniformly labeled internal standards for twelve mycotoxins, and extraction using 50% acetonitrile. Homogeneity of prepared samples, defined as particle size Dv90 < 850 µm for the tested matrices, was characterized using a laser diffraction particle size analyzer, and reached using cryogenic grinding procedures. The majority of recoveries in the four matrices for aflatoxins and ochratoxin A spiked at 1–100 ng/g; fumonisins, T-2 toxin, HT-2 toxin, and zearalenone spiked at 10–1000 ng/g, ranged from 80 to 120% with relative standard deviations (RSDs) of <20%. Deoxynivalenol was not detected at 10 and 100 ng/g in plums, and additional troubleshooting procedures using liquid-liquid extraction (LLE), solid phase extraction (SPE), and elution gradient were evaluated to improve the detectability of the mycotoxin. Furthermore, we confirmed the identity of detected mycotoxins, ochratoxin A and deoxynivalenol, in incurred samples using enhanced product ion scans and spectral library matching.

Determination of lipophilic marine biotoxins in shellfish foods by ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry

An ultra performance liquid chromatography-hybrid triple quadrupole linear ion trap-mass spectrometry(UPLC-QqLIT-MS) was established for determination of lipophilic marine biotoxins in shellfish. And the 12 lipophilic marine biotoxins in shellfish were surveyed. The lipophilic marine biotoxins in homogenized shellfish were ultrasonically extracted by methanol in super-sonic instrument, and cleaned up by solid phase extraction of Strata-X column, and eluted with methanol(containing 0.3% ammonia water). The elution was diluted with water, and cleaned by 0.22 μm millipore filter. The filtrate was separated on a Waters ACQUITY UPLC BEH C_(18) column(150 mm×2.1 mm, 1.7 μm)by gradient elution in 12 minutes with acetronitrile-water(containing 0.01%(V/V) ammonia and 2 mmol/L ammonium formate) as mobile phase, and detected by ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS), identified by electrospray ionization(ESI) in simultaneous scanning mode of positive and negative ions using multiple reaction monitoring, and quantified with external standards. Information dependent acquisition scan function(IDA) combined with enhanced production scan(EPI) was used to confirm the 12 lipophilic marine biotoxins. The calibration curves of 12 lipophilic marine biotoxins showed good linearity in the range of 0.5-50 μg/L with correlation coefficients were 0.9984-0.9999.The detection limits of the method were 0.15-0.29 μg/kg. The recoveries of three spiking levels ranged from 80.0% to 116.0%, and the relative standard deviation(RSD) were 0.6%-6.4%(n=7). The method for determination of 12 lipophilic marine biotoxins in shellfish by UPLC-QqLIT-MS was of operation convenience, less interference from impurities and good accuracy, which could meet the requirements for the determination of 12 lipophilic marine biotoxins residues in sea foods.

Characterization of drug-related impurities using UPLC-QTrap-MS/MS in three scan modes: Application to daptomycin

A new non-targeted screening strategy was developed for the detection and relative quantification of known and potentially unknown drug-related impurities in the active pharmaceutical ingredient daptomycin using ultra-high performance liquid chromatography, coupled with hybrid triple quadrupole-linear ion trap electrospray ionization mass spectrometry in three different modes. The precursor ion scan mode was first applied using the m/z transitions of 159.2, 313.2, and 341.2 for rapid screening of all daptomycin-related impurities. Precursor ions detected at specific retention times were selected, fragmented, and focused in enhanced product ion scan mode to obtain secondary ion mass spectra and identify drug-related impurities. Finally, each potential compound was relatively quantified in multiple reaction monitoring mode. Repeatability experiments suggest that this impurity profiling method is a reliable strategy for identifying unknown impurities in daptomycin and other drugs.

LC-MS-MS Analysis of Δ9-THC, CBN and CBD in Hair: Investigation of Artifacts.

In forensic toxicology, high-performance liquid chromatography tandem mass spectrometry (LC-MS-MS) is increasingly used for the fast and sensitive measurement of a wide range of drugs. For our routine casework, a LC atmospheric pressure chemical ionization MS-MS method for the quantification of Δ9-tetrahydrocannabinol (Δ9-THC), cannabinol (CBN) and cannabidiol (CBD) in hair was established and fully validated. Separation was achieved using a Kinetex® C18 column (100 mm×2.1 mm, 100 Å, 1.7 μm, Phenomenex) at a flow rate of 0.5 mL/min. Measurements were performed on a QTRAP 5500 mass spectrometer (Sciex, Darmstadt, Germany). Unexpected signals were observed in authentic THC-positive hair samples. First, a signal with a slightly shifted retention time of THC whose origin could be assigned to the isomer Δ8-THC was detected. Second, additional peaks exhibiting the same fragments as CBN and Δ9-THC but eluting at different retention times were detected. Spiking experiments and enhanced product ion scans pointed to the origin of these additional signals as result of in-source decarboxylation of Δ9-tetrahydrocannabinolic acid A (Δ9-THCA-A) into Δ9-THC and further partial oxidation of Δ9-THC into CBN, respectively. Positive findings of Δ9-THCA-A in hair have been shown to derive from external contamination; therefore, the herein described artifacts may be used as indirect markers for external contamination.

Quantification and Confirmation of Zearalenone Using a LC-MS/MS QTRAP System in Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes

A simple, rapid, and efficient liquid chromatography tandem mass spectrometry (LC–MS/MS) method, operated in electrospray ionization (ESI) and quadrupole linear ion trap modes, has been developed for the identification and structural characterization of zearalenone (ZEN) in corn oil. Samples (5 g) were extracted with acetonitrile/water (80:20, v/v). After centrifugation and dilution, the extracts were separated on a C18 analytical column by gradient elution (acetonitrile/water) and analyzed by UPLC–MS/MS. The developed multiple reaction monitoring–information-dependent acquisition–enhanced product ion method enabled quantification and confirmation of the analyte in a single run. Enhanced product ion mode was used for qualitative analysis, while multiple reaction monitoring mode was used for quantitative analysis. An in-house library was constructed for identification. Calibration curve showed good linearity with correlation coefficients (r) higher than 0.995. Limit of detection was determined to be below 0.20 μg kg−1 for ZEN. The recovery for ZEN was in the acceptable range of 86.6 to 97.2%. 82.4 % of the samples were found to contain ZEN among the 51 samples.

Evidence of human metabolites of omeprazole and its structure elucidation by using HPLC-MS

The aim of current study was to identify human phase I metabolites of omeprazole using mass spectrometry. An in-vitro assay was performed to generate metabolites by human liver enzymes. Fragmentation pathway of omeprazole was studied for the structure elucidation of metabolite by MS/MS and MS3 method using previously detected fragments. The base peak of omeprazole with m/z 198 was used for a precursor ion scan to detect possible metabolites containing m/z 198 as a fragment. Additional metabolites should be detected by the loss of a typical neutral group using m/z 148 for the offset of a neutral loss scan. The detected precursor ions (m/z 316, 330, 332, 346, 362) were analyzed by enhanced product ion scan in order to receive a mass spectrum for the comparison with the fragmentation of omeprazole. The three reactions (oxidation, reduction of the Sulphur and demethylation) were observed as phase I reactions.

Application of a fluorous derivatization method for characterization of glutathione-trapped reactive metabolites with liquid chromatography–tandem mass spectrometry analysis

The glutathione (GSH) trapping assay is commonly utilized for the screening and characterization of reactive metabolites produced by drug metabolism. This study describes a fluorous derivatization method for a more sensitive and selective analysis of reactive metabolites trapped by GSH using liquid chromatography-tandem mass spectrometry (LC–MS/MS). In this study, the GSH-trapped reactive metabolites, which were obtained after incubation of the test compounds with human liver microsome (HLM) in the presence of GSH and NADPH, were derivatized using the perfluoroalkylamine reagent through oxazolone chemistry. Since this reaction enabled the selective modification of the α-carboxyl group in GSH, the structural compositions of the metabolites were not affected by the derivatization. Furthermore, the selective analysis of the resulting derivatives could be performed using perfluoroalkyl-modified stationary phase LC separation via the interaction between the perfluoroalkyl-containing compounds, such as fluorous affinity, followed by detection with the precursor ion and/or enhanced product ion scan modes in MS/MS. Finally, we demonstrated the applicability of this method by analyzing perfluoroalkyl derivatives of some drug metabolites trapped by GSH in HLM incubation.

Simultaneous detection of 24 oral antidiabetic drugs and their metabolites in urine by liquid chromatography–tandem mass spectrometry

Drugs are the most frequent cause of hypoglycemia. Though the drug history is usually obvious in diabetic patients, the diagnosis could be a challenge in patients without a history of such exposure. Screening for oral antidiabetic drugs has been recommended as part of the hypoglycemia workup in patients without diabetes. Many published analytical methods of oral antidiabetic agents were usually of limited coverage and restricted to parent drugs only. In the current study, a liquid chromatography–tandem mass spectrometry (LC-MS/MS) analytical system for the simultaneous detection of 24 oral antidiabetic drugs and their metabolites in urine was established and validated. The method covered both conventional as well as the newer antidiabetic drugs such as dipeptidyl peptidase-4 inhibitors and sodium-glucose cotransporter-2 inhibitors. Following sample preparation by solid phase extraction, analytes were detected by LC-MS/MS with multiple reaction monitoring triggered enhanced product ion scan. The method was successfully applied to 233 cases of unexplained hypoglycemia, with 83 oral antidiabetic drugs detected in 51 of the urine samples.

Straightforward Determination of Pyrrolizidine Alkaloids in Honey through Simplified Methanol Extraction (QuPPE) and LC-MS/MS Modes.

Contamination of honey with toxic pyrrolizidine alkaloids (PAs) and their N-oxides (PANOs) was assessed by dilution with acidic methanol and analysis through liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) in three different modes. The hybrid linear trap/triple quadrupole (LC-QTRAP) instrument was used in precursor ion scan (PIS), enhanced product ion scan (EPI), and multiple reaction monitoring (MRM) mode. The parent ions from ions at m/z 120 or 138 amus, characteristic of all the toxic PAs and PANOs in the sample were first scanned by PIS. Then, the presence of each contaminant at specific retention times through its MS2 spectrum was confirmed by EPI. Finally, they were quantified in the MRM mode. The method was validated: recoveries 86–111%, relative standard deviation (RSD) <20%, at 20 and 40 μg/kg, except retrorsine, which showed a RSD of 30% at 20 μg/kg. Honey samples were analyzed and five of them showed levels of 40 μg/kg for the sum (PAs + PANOs). This approach allows the simultaneous determination of PAs and PANOs in honey, even if their chemical standards are not available.

GC–MS and LC–MS/MS workflows for the identification and quantitation of pyrrolizidine alkaloids in plant extracts, a case study: Echium plantagineum

Workflows based on gas and liquid chromatography coupled to mass spectrometry for the identification of toxic pyrrolizidine alkaloids present in plants were developed and applied to Echium plantagineum L., Boraginaceae, extracts. GC–MS based determinations need reduction and derivatization steps prior to MS analysis, which is performed using a Full Scan and Single Ion Monitoring sequence for screening, identification and quantification purposes. The LC–(ESI)–MS/MS determination was performed directly from the extract without derivatization. Acetyl lycopsamine, echimidine, echimidine N-oxide, echiumine, echiumine N-oxide, lycopsamine, lycopsamine N-oxide, 7,9-ditigloylretronecine N-oxide and a not reported PA of m/z 466, were identified and quantified in E. plantagineum extracts, through three operating modes of LC-QTRAP: precursor ion scan, enhanced product ion scan and multiple reaction monitoring. Precursor ion scan detects all the ions that give rise to a daughter ion at m/z 120, the presence of the parent pyrrolizidine alkaloid is confirmed through its MS2 spectrum (enhanced product ion) and quantified by multiple reaction monitoring. These workflows are general approaches to study chemical families using GC/LC-MS. For extracts suspicious of containing pyrrolizidine alkaloids, they are suitable tools for the quality and safety control of food, feed as well as phytotherapeutics.

Quantification and Confirmation of Fifteen Carbamate Pesticide Residues by Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes via LC-MS/MS QTRAP System.

In this research, fifteen carbamate pesticide residues were systematically analyzed by ultra-high performance liquid chromatography–quadrupole-linear ion trap mass spectrometry on a QTRAP 5500 system in both multiple reaction monitoring (MRM) and enhanced product ion (EPI) scan modes. The carbamate pesticide residues were extracted from a variety of samples by QuEChERS method and separated by a popular reverse phase column (Waters BEH C18). Except for the current conformation criteria including selected ion pairs, retention time and relative intensities from MRM scan mode, the presence of carbamate pesticide residues in diverse samples, especially some doubtful cases, could also be confirmed by the matching of carbamate pesticide spectra via EPI scan mode. Moreover, the fragmentation routes of fifteen carbamates were firstly explained based on the mass spectra obtained by a QTRAP system; the characteristic fragment ion from a neutral loss of CH3NCO (−57 Da) could be observed. The limits of detection and quantification for fifteen carbamates were 0.2–2.0 μg kg−1 and 0.5–5.0 μg kg−1, respectively. For the intra- (n = 3) and inter-day (n = 15) precisions, the recoveries of fifteen carbamates from spiked samples ranged from 88.1% to 118.4%, and the coefficients of variation (CVs) were all below 10%. The method was applied to pesticide residues detection in fruit, vegetable and green tea samples taken from local markets, in which carbamates were extensively detected but all below the standard of maximum residue limit.

In vitro and in vivo metabolite identification of a novel benzimidazole compound ZLN005 by liquid chromatography/tandem mass spectrometry.

A novel benzimidazole compound ZLN005 was previously identified as a transcriptional activator of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in certain metabolic tissues. Upregulation of PGC-1α by ZLN005 has been shown to have a beneficial effect in a diabetic mouse model and in a coronary artery disease model in vitro. ZLN005 could also have therapeutic potential in neurodegenerative diseases involving down-regulation of PGC-1α. Given the phenotypic efficacy of ZLN005 in several animal models of human disease, its metabolic profile was investigated to guide the development of novel therapeutics using ZLN005 as the lead compound. ZLN005 was incubated with both rat and human liver microsomes and S9 fractions to identify in vitro metabolites. Urine from rats dosed with ZLN005 was used to identify in vivo metabolites. Extracted metabolites were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using a hybrid linear ion trap triple quadrupole mass spectrometer in full scan, enhanced product ion scan, neutral loss scan and precursor scan modes. Metabolites in plasma and brain of ZLN005-treated rats were also profiled using multiple reaction monitoring. Identified in vitro transformations of ZLN005 include mono- and dihydroxylation, further oxidation to carboxylic acids, and mono-O-glucuronide and sulfate conjugation to hydroxy ZLN005 as well as glutathione conjugation. Identified in vivo metabolites are mainly glucuronide and sulfate conjugates of dihydroxyl, carboxyl, and hydroxy acid of the parent compound. The parent compound as well as several major phase I metabolites were found in rat plasma and brain. Using both in vitro and in vivo methods, we elucidated the metabolic pathway of ZLN005. Phase I metabolites with hydroxylation and carboxylation, as well as phase II metabolites with glucuronide, sulfate and glutathione conjugation, were identified.

Using UHPLC Q-Trap/MS as a complementary technique to in-depth mine UPLC Q-TOF/MS data for identifying modified nucleosides in urine.

Modified nucleosides, metabolites of RNA, are potential biomarkers of cancer before the appearance of morphological abnormalities. It is of great significance to comprehensively detect and identify nucleosides in human urine for discovery of cancer biomarkers. However, the lower abundance, the greater polarity and the matrix effects make it difficult to detect urinary nucleosides. In this paper, an integrated method consisted of sample preparation followed by ultraperformance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC Q-TOF/MS) detection and primary identification, then ultra-high performance liquid chromatography coupled with hybrid triple quadrupole linear ion trap mass spectrometer (UHPLC Q-Trap/MS) further identification and validation were introduced. Firstly, to enrich the nucleosides and eliminate the urine matrix effects, different sorbent materials of solid phase extraction (SPE) and the elution conditions were screened. Secondly, UPLC Q-TOF/MS was used to acquire mass data in MSE mode. The structural formulas of nucleosides in urine sample were primarily identified according to retention time, accurate mass precursor ions and fragment ions from in-house database and online database. Thirdly, the preliminary identified nucleoside structures lacking of characteristic fragment ions were verified by UHPLC Q-Trap/MS in multiple reaction monitoring trigger enhanced product ion scan (MRM-EPI) and neutral loss scan (NL). At last, phenylboronic acid (PBA)-based SPE was utilized due to its higher MS signal and weaker matrix effects under optimized extraction conditions. Fifty-five nucleosides were primarily identified by UPLC Q-TOF/MS, among which 50 nucleosides were confirmed by UHPLC Q-Trap/MS. Five nucleosides, namely 4',5'-didehydro-5'-deoxyadenosine, 4',5'-didehydro-5'-deoxyinosine, isonicotinamide riboside, peroxywybutosine and hydroxywybutosine, were found from urine for the first time. The results will expand the Human Metabolome Database (HMDB).

Comparison of the Multiple Reaction Monitoring and Enhanced Product Ion Scan Modes for Confirmation of Stilbenes in Bovine Urine Samples Using LC-MS/MS QTRAP® System.

In accordance with Commission Decision 2002/657/EC, confirmatory methods for the detection of prohibited substances should comply with specific requirements, including the criteria for confirmation. Two strategies: multiple reaction monitoring (MRM) and enhanced product ion (EPI) scanning functions were compared for confirming the anabolic compounds from synthetic stilbenes group in bovine urine samples. In the research, twenty samples fortified at the Recommended Concentration (RC) of 1 µg L−1 with diethylstilbestrol, dienestrol and hexestrol were analyzed by liquid chromatography-tandem mass spectrometry on a QTRAP 5500 instrument. The analytical procedure, validated in accordance with the Commission Decision 2002/657/EC, used in the official control of hormones in Poland was applied. The validation parameters were in agreement with 2002/657/EC performance criteria. The effectiveness of MRM and EPI scanning modes for confirmation purposes was evaluated based on the percentage of the results confirmed. In all urine samples recorded in the MRM mode, the confirmation criteria (retention time, relative intensities between transitions) have been fulfilled. The presence of stilbenes in all urine samples using EPI scan mode was confirmed too as evidenced by a good matching of stilbenes spectra in the samples to the reference spectra with critical match factor above 0.7. The results of the research show that EPI scanning function provides the same effectiveness for confirmation of banned compounds as the mostly used MRM scan mode and can be an additional tool to confirm the doubtful case results in the analysis of hormones residues, even at such low concentration levels.

Metabolism and bioactivation of the tricyclic antidepressant amitriptyline in human liver microsomes and human urine.

Amitriptyline is a widely used tricyclic antidepressant, but the metabolic studies were conducted almost 20 years ago using high-performance liquid chromatography coupled with ultraviolet detector or radiolabeled methods. First, multiple ion monitoring (MIM)- enhanced product ion (EPI) scan was used to obtain the diagnostic ions or neutral losses in human liver microsome incubations with amitriptyline. Subsequently, predicted multiple reaction monitoring (MRM)-EPI scan was used to identify the metabolites in human urine with the diagnostic ions or neutral losses. Finally, product ion filtering and neutral loss filtering were used as the data mining tools to screen metabolites. Consequently, a total of 28 metabolites were identified in human urine after an oral administration using LC-MS/MS. An integrated workflow using LC-MS/MS was developed to comprehensively profile the metabolites of amitriptyline in human urine, in which five N-acetyl-l-cysteine conjugates were characterized as tentative biomarkers for idiosyncratic toxicity.

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.

Rapid, automated online SPE-LC-QTRAP-MS/MS method for the simultaneous analysis of 14 phthalate metabolites and 5 bisphenol analogues in human urine

Phthalates and bisphenol A (BPA) have received special attention in recent years due to their frequent use in consumer products and potential for adverse effects on human health. BPA is being replaced with a number of alternatives, including bisphenol S, bisphenol B, bisphenol F and bisphenol AF. These bisphenol analogues have similar potential for adverse health effects, but studies on human exposure are limited. Accurate measurement of multiple contaminants is important for estimating exposure. This paper describes a sensitive and automated method for the simultaneous determination of 14 phthalate metabolites, BPA and four bisphenol analogues in urine using online solid phase extraction coupled with high-performance liquid chromatography/tandem mass spectrometry using a hybrid triple-quadrupole linear ion trap mass spectrometer (LC-QTRAP-MS/MS), requiring very little sample volume (50µL). Quantification was performed under selected reaction monitoring (SRM) mode with negative electrospray ionization. The use of SRM combined with an enhanced product ion scan within the same analysis was examined. Unequivocal identification was provided by the acquisition of three SRM transitions per compound and isotope dilution. The analytical performance of the method was evaluated in synthetic and human urine. Linearity of response over three orders of magnitude was demonstrated for all of the compounds (R2>0.99), with method detection limits of 0.01–0.5ng/mL and limits of reporting of 0.07–3.1ng/mL. Accuracy ranged from 93% to 113% and inter- and intra-day precision were <22%. Finally, the validated method has been successfully applied to a cohort of pregnant women to measure biomarker concentrations of phthalates and bisphenols, with median concentrations ranging from 0.3ng/mL (bisphenol S) to 18.5ng/mL (monoethyl phthalate).

Identification and Characterization of Kukoamine Metabolites by Multiple Ion Monitoring Triggered Enhanced Product Ion Scan Method with a Triple-Quadruple Linear Ion Trap Mass Spectrometer.

Kukoamines are a series of bioactive phytochemicals conjugated by a polyamine backbone and phenolic moieties. Understanding the structural diversity of kukoamine metabolites in plants is meaningful for drug discovery. In this study, an LC-MS/MS method was established for kukoamine profiling and characterization from lycii cortex (LyC) via a triple-quadrupole linear ion trap mass spectrometry (Q-TRAP). On the basis of the typical fragmentation of kukoamine, a diagnostic ion, which represents the features of the backbone and phenolic substitute, was chosen as the product ion for precursor ion scan, and then the screened precursor ions were applied to a successive multiple ion monitoring triggered enhanced product ion scan (MIM-EPI) to simultaneously present the profile survey and MS/MS acquisition. Because the MIM narrowed the ion scan range in Q1 and the ion trap enhanced the ion fragments passing through Q2, the qualitative capability of quadrupole MS can be greatly improved, especially for capture of the uncommon metabolites. There are 12 kukoamine metabolites identified from LyC, with either spermine or spermidine backbone and with conjugation of one to three dihydrocaffeoyls or other kinds of phenolic moieties. Except for kukoamines A and B, other metabolites were identified in LyC for the first time. This approach can be utilized for metabolite identification in other substrates.