Triple Quad Mass Spectrometry Research Articles

LC-MS/MS-based metabolite quantitation of the antiviral prodrug baloxavir marboxil, a new therapy for acute uncomplicated influenza, in human plasma: Application to a human pharmacokinetic study

Baloxavir marboxil (BXM) is a novel orally administrated prodrug for the treatment of acute uncomplicated influenza. In the present study, a bioanalytical LC-MS/MS method was developed and validated for the quantification of baloxavir acid (BXA), the active form of baloxavir marboxil in plasma of healthy volunteers using dolutegravir as an internal standard (IS) following plasma protein precipitation with acetonitrile. BXA and the internal standard were chromatographically separated using Waters Xterra® MS C8 column (5 µm, 4.6 × 50 mm) and a mobile phase comprised of 10.0 mM ammonium formate pH 3.5 and acetonitrile (80:20, v/v) delivered at a flow rate of 0.6 mL/min. The transitions of m/z 484.00 → 247.0 and 420.30 → 277.1 for BXA and IS, respectively in multiple reaction monitoring (MRM) mode in a positive ESI interface were used for quantitation through triple-quad mass spectrometry, API 4000. The method linearity was proven across the concentration range of 0.5–200.0 ng/mL, adjusted, and validated completely in accordance with the bioanalytical guidelines of the United States-FDA. Finally, the present method was effectively applied for the pharmacokinetic study of BXA in healthy human volunteers with accepted reproducibility and ruggedness.

Optimization and validation of a QuEChERS-based method for the simultaneous environmental monitoring of 218 pesticide residues in clay loam soil

A modified QuEChERS method was optimized, validated and verified for the extraction of 218 pesticide residues in agricultural soil samples. The 218 analytes are extracted using a single step, without clean-up, with matrix-matched calibration, and two complementary techniques: liquid and gas chromatography tandem triple quad mass spectrometry (LC-MS/MS and GC-MS/MS). Some of the parameters such as salts, acidity of the extraction solvent, sample moisture and some mechanical changes in the procedure were optimized to improve the overall performance for the target compounds and the soil matrix. The method was fully validated on a representative agricultural soil sample of the Canary Islands (clay loam soil) in terms of linearity, accuracy and precision. To avoid matrix effects, matrix-matched calibration curves (R2 ≥ 0.99) were used for all target analytes. 100% of the compounds can be quantified with limits of quantification (LOQ) lower than the limit typically used in soils (50 ng g−1), with 92% of compounds presenting a LOQ that is at least 10 times lower than that normally required. The limits of detection (LOD) ranged between 0.024 and 6.25 ng g−1. The validated method was applied to a series of actual samples of agricultural soil (n = 18). In addition, as a further verification of its potential, the results of the application of the method in the investigation of clay loam soil samples that were obtained from underneath wildlife carcasses in the context of an environmental forensic investigation are also presented.

Back Cover: Enantiomeric separation and estimation of stiripentol by liquid chromatography mass spectrometer in human plasma

DOI: 10.1002/sscp.202000034 The cover picture shows achiral Stiripentol that was to be extracted from the human plasma for a bioequivalence study for a test and a reference product of 500 mg dose. The enantiomers, R and S Stiripentol behave differently in the human body, hence it was necessary to first extract them from human plasma and then separate them using ultra-fast liquid chromatography (UFLC) and chiral chromatography. High sensitivity for the method was achieved by using triple quad mass spectrometry in unit resolution mode. All these ideas put together resulted in a fantastic enantiomeric resolution with a complete base to base separation for Stiripentol with a resolution factor of more than 4.

Liquid chromatography-diode array-mass spectrometric analysis of amino and mercapto compounds coupled with chloroimino derivatization reagent

A new derivatization reagent, N-(naphthalen-1-yl)-2-oxopropanehydrazonoyl chloride (UOSA54), was prepared and coupled with four drugs, bearing primary amino, secondary amino or mercapto functional groups. Glucosamine sulfate (GLU), cysteine (CYS), captopril (CAP), and vildagliptin (VIL), were used as representative reactive analytes. The prepared reagent was successfully coupled with the targeted analytes in the presence of triethylamine (TEA) as hydrochloride acceptor and acetonitrile as solvent. The resulting reaction products were separated by high-performance liquid chromatography and monitored simultaneously by diode array and triple quad mass spectrometry detectors. Enhanced DAD and electrospray ionization-MS (ESI-MS) responses were observed for the derivatized products. Complete derivatization of VIL was achieved after heating at 65 ± 3 °C for 4 min, while other analytes were derivatized instantaneously at room temperature. Both, the ESI-ionization suppression, due to the excess reagent, and matrix effect, due to co-eluted biogenic plasma constituents, were negligible. The derivatized GLU, CYS, CAP, and VIL showed a maximum absorption wavelength at 376, 417, 340, and 376 nm, with MS-limit of quantification value of 250.0, 2.0, 2.5, and 3.0 pg/μL, respectively. The relative ESI-MS response of UOSA54 derivatization products was within the range of 0.6–4.1 compared with dansylated products. The method was optimized and validated for optimal reaction product stability, sensitivity, linearity, range, precision, and accuracy. The percentage recovery was exceeding 97.2%, with an RSD value of less than 4.0%. The limit of quantification of targeted analytes was ranged from 80.0 to 0.7 pg/μL.

Simultaneous determination of seven effective components of Tripterygium glycosides in human biological matrices by ultra performance liquid chromatography–triple quadrupole mass spectrometry

This paper developed a novel, sensitive, and selective ultra-performance liquid chromatography–triple quad mass spectrometry method to simultaneously determine seven effective constituents (triptolide, triptophenolide, celastrol, wilforgine, wilforine, wilfordine and wilfortrine) of Tripterygium glycosides (GTW) in human serum and urine. The chromatographic separation was performed on the C18 column using an ammonium acetate buffer solution–acetonitrile (both containing 0.1% formic acid) in a gradient program with a flow rate of 0.3 mL/min. Monitoring reaction mode was applied to target compounds quantitative analysis in the positive electrospray ionization (ESI) mode. The analysis process took 11 min in total. This method was fully validated with a linear range of 1–200 ng/mL for triptolide, 0.4–80 ng/mL for celastrol, 0.1–20 ng/mL for triptophenolide, wilforgine, wilforine, wilfordine, and wilfortrine. The intra-day and inter-day accuracy and precision of the target compounds all met the 15% criterion in both serum and urine. Extraction recovery, matrix effect, and dilution integrity were also validated. The short-term and long-term stability results indicated that all the constituents were stable in human serum and urine under the investigated storage conditions. 10 patients' specimens were collected and analyzed. Most of the compounds exhibited the tendency of higher concentration in urine than that in serum. The concentration that was detected in the serum and in the urine of alkaloids showed a positive-correlation property. This is the first time that triptophenolide was quantified in human bio-matrices. The method is feasible for multi-components therapeutic monitoring or pharmacokinetics study in clinical pharmaceutical research of Tripterygium glycosides.

Sulfur determination in concrete samples using laser-induced breakdown spectroscopy and limestone standards

A LIBS equipment operating at 532nm was optimized and used for sulfur determination in concrete samples. The influence of He atmosphere in a gas-tight chamber (1000–200mbar) on S I 921.29nm line sensitivity, signal-to-background and signal-to-noise ratio was studied at gate delays 100–2000ns. Wide range of gate delays from 500 to about 1000ns and pressures from several hundreds of mbar to the atmospheric pressure can be used for the desired detection of sulfur. The LIBS quantification was done using a simple calibration method. A synthetic limestone enriched by defined amounts of sodium sulfate was newly employed for direct quantification of S in concrete. This powder material was pressed into pellets and ablated with the LIBS system. The average content of sulfur as SO3 in the samples was 0.41–0.70wt% by LIBS and 0.43–0.61wt% by a reference standard procedure employing gravimetry and Inductively Coupled Plasma Triple Quad Mass Spectrometry (ICP-QQQMS). The uncertainty of the yielded LIBS results covers also the dispersion of the points in the calibration line and ranges from 16 to 28% at the probability level of 95%. The uncertainty of the ICP-QQQMS results was almost 10%. No correction on different signal response on the limestone and on the concrete was necessary.

Compositional and antioxidant properties of anthocyanin-rich products prepared from purple wheat

Demands for anthocyanin-rich ingredients are steadily growing due to their positive functions in human health and food coloration. Commercially available purple wheat was evaluated as a source of functional ingredients based on anthocyanin composition and antioxidant properties. Anthocyanins were concentrated in the bran fraction by about 2-fold and then the bran was further processed into anthocyanin-rich powder using ethanol extraction and column purification. Batch studies showed that anthocyanin concentration factor increased by about 81- to 135-fold in the powder depending on batch size. Characterization of anthocyanins with a triple quad mass spectrometry showed a complex anthocyanin profile with 5 pigments making up 93–96% of the total anthocyanin content. These pigments were cyanidin-3-glucoside, cyanidin-3-(6-malonyl glucoside), cyanidin-3-rutinoside, peonidin-3-glucoside and peonidin-3-(6-malonylglucoside) but their concentrations varied between bran and powder. The purple wheat bran and powder exhibited exceptional antioxidant capacity based on scavenging of DPPH, ABTS and peroxyl radicals holding a promise as functional ingredients.

Abstract 4647: Mass spectrometric analysis of oncometabolite 2-hydroxyglutarate in clinical gliomas with the isocitrate dehydrogenase-1 mutation

Abstract Introduction: Mutations of the isocitrate dehydrogenase (IDH)-1/2 genes frequently occur in certain malignancies, including gliomas. Mutant IDH-1/2 proteins gain a new ability to produce oncometabolite 2-hydroxiglutarate (2HG), which results in a different clinical impact on gliomas in comparison to those with a wild-type IDH. The status of IDH mutations has been determined by immunohistochemistry (IHC) using the IDH1-R132H antibody or through the DNA sequencing of IDH1-R132C/R132S; however, its practical applications are still under discussion, and the significance of the oncometabolite 2HG evaluation is not clear. In this study, we analyzed the serum and tissue levels of 2HG in clinical gliomas using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the matrix assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) system. Method: Glioma samples (n = 78, matched pairs of serum and flash frozen tissue) were obtained between October 2007 to January 2015 at the National Cancer Center Hospital, Japan. High performance liquid chromatography (Nexera X2, Shimadzu) and triple quad mass spectrometry (QTRAP4500, AB SCIEX) were used for LC-MS/MS analysis. The two MALDI-MSI systems, quadrupole ion trap time-to-flight mass spectrometry imaging (iMScope, Shimadzu), and high-resolution atmospheric pressure mass spectrometry imaging (AP-SMALDI, TransMIT; Q-Exactive, Thermo Fisher Scientific) were used for analyzing the 2HG tissue distribution after coating with 9-aminoacridine or 2,5-dihydroxybenzoic acid. The IDH mutation was determined by direct sequencing and IHC. Results: The median 2HG concentration in serum was 21.43 ng/mL (range: 9.58 to 62.51) in this study, and no significant correlation was observed between the IDH mutation and the wild type status (mutation, n = 28, median 20.2 ng/mL, range 9.58 to 62.51 vs. wild type, n = 50, median 22.88 ng/mL, range 11.51 to 40.32; Wilcoxon test p = 0.1597). The median of the 2HG tissue concentration in the entire group was 0.16 ng/μg, and a significant difference was seen between the IDH mutant and wild type groups (mutation, median 4.86 ng/μg, range 0.51 to 49.86 vs. wild type, median 0.09 ng/μg, range 0.02 to 0.45; Wilcoxon test p = <0.0001). In the MALDI-MSI analysis, the average 2HG ion intensities were 381.62/mm2 in the IDH mutation and 8.25/mm2 in the wild-type IDH tissues (Wilcoxon test p = 0.016), which showed a positive correlation with the 2HG tissue concentration by LC-MSMS analyses (Spearman ρ = 0.87, p = 0.0003). The MSI results indicate a possible link between the 2HG tissue distributions and the histology of IDH mutant sections. Conclusion: Tissue 2HG measurement using mass spectrometric analyses may be a useful alternative in determining IDH mutations. Further research may provide a biological perspective and help gain some insights on the clinical significance of 2HG. Citation Format: Mitsuhiro Hayashi, Hiroaki Aikawa, Makoto Ohno, Koichi Ichimura, Yuko Matsushita, Mayu Ohuchi, Mariko Mizui, Akihiko Yoshida, Yoshitaka Narita, Akinobu Hamada. Mass spectrometric analysis of oncometabolite 2-hydroxyglutarate in clinical gliomas with the isocitrate dehydrogenase-1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4647. doi:10.1158/1538-7445.AM2017-4647

Studies on Isotope Ratio Measurement of Cl by Inductively Coupled Plasma Triple-quad Mass Spectrometry.

Fundamental studies on isotope ratio measurement of Cl were carried out using inductively coupled plasma triple-quad mass spectrometry (ICP-MS/MS) and the analytical performance obtained was compared to that obtained by ICP sector field mass spectrometer (ICP-SFMS). Though the polyatomic ion interferences of 16O18O1H and 36Ar1H with respect to 35Cl and 37Cl, respectively, made a negative effect on the accuracy and the precision for isotope ratio measurements of Cl, the ICP-SFMS could eliminate these interferences by medium mass resolution mode (m/Δm = 4000) and achieved the isotope ratio measurements with 0.2 - 0.5% of relative standard deviation (RSD) at the concentrations of Cl from 1 to 10 mg kg-1. In the case of ICP-MS/MS, both the single-MS mode without collision reaction gas and the MS/MS mode with collision reaction gases such as oxygen (O2) and hydrogen (H2) were examined and compared their analytical sensitivities as well as the precisions of isotope ratio measurement of Cl. The precisions of Cl isotope ratio measurements were 3 - 14% of RSD at the concentrations of Cl from 5 to 100 mg kg-1, when single-MS mode was carried out, even though the similar isotope ratios of 35Cl/37Cl could be obtained. In the case of O2 gas for MS/MS mode with mass-shift method, precisions of 0.3 - 2% of RSD were obtained at the concentration range of 1 - 100 mg kg-1. In the case of H2 gas, similar sensitivities as those obtained by ICP-SFMS and the precisions of 0.2 - 0.5% of RSD at the concentration range of 1 - 10 mg kg-1 were obtained. From these results, it was evaluated that the ICP-MS/MS in MS/MS mode with collision reaction gas could be used for Cl isotope ratio measurements for such studies as stable isotope tracers, isotope abundance measurements in nuclear chemistry and accurate determinations by isotope dilution mass spectrometry.

Pharmacokinetics of five ginsenosides of Shexiang Baoxin pill in plasma of myocardial infarction rats

Shexiang Baoxin pill(SBP) is widely used for treating coronary heart disease in clinic, with ginsenosides as its main effective component. This study was designed to investigate and compare the pharmacokinetic characteristics of five ginsenosides of five compounds after multiple oral administrations, ginseng extract(GE) and SBP in myocardial infarction rats. After intragastric administration to myocardial infarction rats, the plasma samples were analyzed by liquid chromatography tandem triple-quad mass spectrometry. The results showed that Cmax of five compounds in all groups were less than 200 μg•L⁻¹. Tmax of corresponding analytes between groups revealed that ginsenosides in both SBP and GE were absorbed faster than each of the five compounds, indicating that GE and compounds contain components promoting absorption of ginsenosides. The oral administration doses of ginsenosides in SBP were the least in all groups, but with the greatest AUC0-tand AUCINF, which indicated that ginsenosides in SBP had the best absorption in all groups after oral administration to myocardial infarction rats. This study also demonstrated that compound is the best form of traditional Chinese medicine.

Increased catabolism and decreased unsaturation of ganglioside in patients with inflammatory bowel disease.

To investigate whether accelerated catabolism of ganglioside and decreased ganglioside content contribute to the etiology of pro-inflammatory intestinal disease. Intestinal mucosa from terminal ileum or colon was obtained from patients with ulcerative colitis or inflammatory Crohn's disease (n = 11) undergoing bowel resection and compared to control samples of normal intestine from patients with benign colon polyps (n = 6) and colorectal cancer (n = 12) in this observational case-control study. Gangliosides and phospholipids of intestinal mucosa were characterized by class and ceramide or fatty acid composition using liquid chromatography triple-quad mass spectrometry. Content and composition of ganglioside classes GM1, GM3, GD3, GD1a, GT1 and GT3 were compared among subject groups. Content and composition of phospholipid classes phosphatidylcholine (PC) and phosphatidylethanolamine were compared among subject groups. Unsaturation index of individual ganglioside and phospholipid classes was computed and compared among subject groups. Ganglioside catabolism enzymes beta-hexosaminidase A (HEXA) and sialidase-3 (NEU3) were measured in intestinal mucosa using western blot and compared among subject groups. Relative GM3 ganglioside content was 2-fold higher (P < 0.05) in intestine from patients with inflammatory bowel disease (IBD) compared to control intestine. The quantity of GM3 and ratio of GM3/GD3 was also higher in IBD intestine than control tissue (P < 0.05). Control intestine exhibited 3-fold higher (P < 0.01) relative GD1a ganglioside content than IBD intestine. GD3 and GD1a species of ganglioside containing three unsaturated bonds were present in control intestine, but were not detected in IBD intestine. The relative content of PC containing more than two unsaturated bonds was 30% lower in IBD intestine than control intestine (P < 0.05). The relative content of HEXA in IBD intestine was increased 1.7-fold (P < 0.05) and NEU3 was increased 8.3-fold (P < 0.01) compared to normal intestine. Intestinal mucosa in IBD is characterized by increased GM3 content, decreased GD1a, and a reduction in polyunsaturated fatty acid constituents in GD3, GD1a and PC. This study suggests a new paradigm by proposing that IBD occurs as a consequence of increased metabolism of specific gangliosides.

Possibilities for discrimination between chewing of coca leaves and abuse of cocaine by hair analysis including hygrine, cuscohygrine, cinnamoylcocaine and cocaine metabolite/cocaine ratios.

Contrary to the illegal use of any form of manufactured cocaine, chewing of coca leaves and drinking of coca tea are allowed and are very common and socially integrated in several South American countries. Because of this different legal state, an analytical method for discrimination between use of coca leaves and abuse of processed cocaine preparations is required. In this study, the applicability of hair analysis for this purpose was examined. Hair samples from 26 Argentinean coca chewers and 22 German cocaine users were analysed for cocaine (COC), norcocaine (NC), benzoylecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), cinnamoylcocaine (CIN), tropacocaine (TRO), cuscohygrine (CUS) and hygrine (HYG) by hydrophilic interaction liquid chromatography (HILIC) in combination with triplequad mass spectrometry (MS/MS) and hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS). The following concentrations (range, median, ng/mg) were determined in hair of the coca chewers: COC 0.085-75.5, 17.0; NC 0.03-1.15, 0.12; BE 0.046-35.5, 6.1; EME 0.014-6.0, 0.66; CE 0.00-13.8, 0.38; CIN 0.005-16.8, 0.79; TRO 0.02-0.16, 0.023; CUS 0.026-26.7, 0.31. In lack of a reference substance, only qualitative data were obtained for HYG, and two metabolites of CUS were detected which were not found in hair of the cocaine users. For interpretation, the concentrations of the metabolites and of the coca alkaloids in relation to cocaine were statistically compared between coca chewers and cocaine users. By analysis of variance (ANOVA) significant differences were found for all analytes (α = 0.000 to 0.030) with the exception of TRO (α = 0.218). The ratios CUS/COC, CIN/COC and EME/COC appeared to be the most suitable criteria for discrimination between both groups with the means and medians 5-fold to 10-fold higher for coca chewers and a low overlap of the ranges between both groups. The same was qualitatively found for HYG. However, these criteria cannot exclude cocaine use in addition to coca chewing. In this regard screening for typical cutting agents can be helpful and led to the detection of levamisole (21×), lidocaine (6×) and paracetamol (3×) in the 22 samples from German cocaine users, whereas no levamisole, lidocaine (3×) and paracetamol (1×) were found in hair from the Argentinean coca chewers. These criteria have to be confirmed for South American cocaine consumers including smokers of coca paste and may be different because of different composition of the drug and other use habits.

O11: Differentiation between chewing of coca leaves and abuse of cocaine by hair analysis

Aims Contrary to the illegal use of any form of manufactured cocaine, chewing of coca leaves and drinking of coca tea is allowed and is very common and socially integrated in several South American countries. Because of this different legal state, an analytical method for discrimination between use of coca leaves and abuse of artificial cocaine preparations is required. In this study, the applicability of hair analysis for this purpose was examined. Methods Hair samples from 26 Argentinean coca chewers and 22 German cocaine users were analysed for cocaine (COC), norcocaine (NC), benzoylecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), cinnamoylcocaine (CIN), tropacocaine (TRO), cuscohygrine (CUS) and hygrine (HYG) by hydrophilic interaction liquid chromatography (HILIC) in combination with triplequad mass spectrometry (QQQ-MS) and hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS). Results The following concentrations (range, mean, median, ng/mg) were determined in hair of the coca chewers: COC 0.085–75.5, 22.7, 17.0; NC 0.03–1.15, 0.27, 0.12; BE 0.046–35.5, 10.1, 6.1; EME 0.014–6.0, 1.04, 0.66; CE 0.00–13.8, 3.06, 0.38; CIN 0.005–16.8, 2.82, 0.79; TRO 0.02–0.16, 0.035, 0.023; CUS 0.026–26.7, 2.45, 0.31. In lack of a reference substance, only qualitative data were obtained for HYG and two metabolites of CUS were detected which were not found in hair of the cocaine users. For interpretation, the concentrations of the metabolites and the coca alkaloids in relation to cocaine were statistically compared between coca chewers and cocaine users. By analysis of variance (ANOVA) significant differences were found for all analytes (α = 0.000 to 0.030) with the exception of TRO (α = 0.218). The ratios CUS/COC, CIN/COC and EME/COC appeared to be the most suitable criteria for discrimination between both groups with the means and medians 5-fold to 10-fold higher for coca chewers and a low overlap of the ranges between both groups. The same was qualitatively found for HYG. Screening for typical cutting agents led to detection of levamisole (21x), lidocaine (6x) and paracetamol (3x) in the 22 samples from German cocaine users whereas no levamisole, lidocaine (3x) and paracetamol (1x) was found in hair from the Argentinean coca chewers. Conclusion Hair analysis proved to be a suitable tool for discrimination between chewing of coca leaves and use of manufactured cocaine by detection of other coca alkaloids and their concentrations relative to cocaine, and by the absence of typical adulterants. However, these criteria have to be confirmed for South American cocaine consumers including smokers of coca paste because of possible different composition of illegal cocaine preparations and other use habits. They cannot exclude occasional cocaine use in addition to chewing of coca leaves.

The utility of fluid biomarkers in research, trial design and clinical practice

An optimistic and logical conclusion from recent clinical trials on AD disease-modifying drugs is that anti-Aβ compounds will be effective if therapy is initiated in a very early stage of the disease, before neurodegeneration is too severe. Therefore, we need sensitive and reliable tools to enable diagnosis of prodromal AD. Luckily, research advances have provided the field with both imaging (MRI and PET) and cerebrospinal fluid (CSF) biomarkers for this purpose. The CSF biomarkers total tau (reflecting neuronal degeneration), Aβ42 (reflecting Aβ deposition in plaques), and phosphorylated tau (probably related to tangle formation), have high diagnostic accuracy for AD dementia and can differentiate AD from normal aging and several important differential diagnoses. Large multi-center studies also show that that these CSF biomarkers can identify prodromal AD cases in MCI cohorts To enable a general introduction of CSF biomarkers in clinical routine, several standardization initiatives have been initiated by the Alzheimer's Association, including the Global Biomarker Standardization Consortium (GBSC) and the world-wide Quality Control (QC) program for CSF biomarkers. These efforts include establishing a reference method for CSF Aβ42 based on SRM Triple-Quad mass spectrometry. The interest in using biomarkers to study AD pathogenesis directly in man has increased. Some studies have evaluated CSF biomarkers in normal elderly to identify when in the preclinical phase of AD the biomarkers change from normal to positive and thereby which pathogenic process comes first in the disease course. Data so far largely come from single-LP cross-sectional studies with either clinical follow-up or extrapolation to symptoms onset, which leaves more than one option for interpretation of results. Last, CSF biomarkers may be valuable to monitor the biochemical effect of novel drugs directly in living AD patients. Results from early-phase BACE1 inhibitor trials indicate that CSF biomarkers (Aβ and sAPP isoforms) show the expected response to treatment. Such evidence of target engagement in man may be important to make the decision to proceed to large clinical trials. In addition, evidence from downstream biomarkers, such as T-tau and P-tau, may together with a favourable effect on cognition be essential for labelling a drug as disease-modifying.

Optimized extraction method for LC–MS determination of bisphenol A, melamine and di(2-ethylhexyl) phthalate in selected soft drinks, syringes, and milk powder

Described below is an optimized solid-phase extraction method (SPE) for the simultaneous determination of three hazardous plastic additives. Two high-performance liquid chromatographic-electrospray ionization-mass spectrometric (HPLC-ESI-MS) methods were developed and validated to estimate the melamine (MEL), bis(2-ethylhexyl) phthalate (DEHP), and bisphenol A (BPA) contents in drinking water, syringes, soft drinks, and dry milk powder. One extraction procedure optimally recovered all three substances from the different matrices. Two extraction columns were combined and included a silica gel LiChroprep RP-2 column (20:1, g/g, top column) and a Sep-Pak with a C18 column (500mg, bottom column). The analytical column was an Agilent Eclipse XDB-C8 column, 4.6mm×150mm, 5μm, maintained at 50±2°C. MEL and DEHP were monitored by positive triple quad mass spectrometry (TQ-MS) using an acidic mobile phase, while BPA was monitored by negative TQ-MS using a mobile phase containing a 0.05% ammonia solution. The general linear range of the three compounds ranged from 12 to 1000pg/μL in the injected solution (25μL). The average extraction recoveries were within the range of 83.0-102.5%. Relatively high concentrations of BPA and DEHP were found in the milk powder and sterile syringes.

Systematic qualitative and quantitative assessment of anthocyanins, flavones and flavonols in the petals of 108 lotus (Nelumbo nucifera) cultivars

Petal colour is one of the major characteristics that determine the ornamental value of lotus. To assess the contribution of different flavonoids to this character, composition and content of anthocyanins, flavones and flavonols were analysed through high performance liquid chromatography coupled with photodiode array detection tandem electrospray ionisation triple quad mass spectrometry in 108 lotus cultivars with red, pink, yellow, white and red/white pied petal colours. Totally, five anthocyanins and fourteen flavones and flavonols were detected and quantified. In general, the yellow, white and pied species hardly contained any anthocyanins; red cultivars contain more than pink cultivars. Among the five anthocyanins, malvidin 3-O-glucoside was the most abundant one in all the cultivars that contain anthocyanin. The fourteen flavones and flavonols belonged to four groups based on their aglycones. Except for the yellow cultivars, kaempferol-derivatives were the most abundant one. These data might be helpful in lotus breeding for different colours.

Quantification of the Maillard reaction product 6-(2-formyl-1-pyrrolyl)-l-norleucine (formyline) in food

The Maillard reaction product 6-(2-formyl-1-pyrrolyl)-l-norleucine (formyline) was recently identified as a new pyrrole amino acid resulting from the reaction of free and protein-bound lysine residues with the 1,2-dicarbonyl compound 3-deoxypentosone, pentose sugars, and degradation products of disaccharides. In the present work, simultaneous analysis of formyline and its structural analog, pyrraline, in enzymatically hydrolyzed food matrices was achieved on an RP-phenyl column by high-pressure liquid chromatography (HPLC) with UV detection. The presence of formyline was for the first time unequivocally verified in the hydrolyzates and standard samples by HPLC coupled to triple quad mass spectrometry due to identity of product ion spectra after targeted fragmentation of the molecular ion of formyline. A total of 68 food samples were analyzed. Low concentrations of formyline were detected in milk and whey products. In breakfast cereals, pasta, and bakery products, formyline was quantified in concentrations up to 34.8 mg/kg. In the outmost bread crust, 0.6 % of lysine residues can be modified to formyline. Moreover, it was shown by analysis of concentrated beer samples that both formyline and pyrraline are constituents of beer proteins. From our data, we conclude that at least 2–3 mg of formyline and 20–40 mg of pyrraline are ingested with the daily diet.

Simultaneous qualitative assessment and quantitative analysis of flavonoids in various tissues of lotus (Nelumbo nucifera) using high performance liquid chromatography coupled with triple quad mass spectrometry

Flavonoid composition and concentration were investigated in 12 different tissues of ‘Ti-1’ lotus (Nelumbo nucifera) by high performance liquid chromatography equipped with photodiode array detection tandem electrospray ionization mass spectrometry (HPLC-DAD-ESI-MSn). A total of 20 flavonoids belonging to six groups (myricetin, quercetin, kaempferol, isohamnetin, diosmetin derivatives) were separated and identified. Myricetin 3-O-galactoside, myricetin 3-O-glucuronide, isorhamnetin 3-O-glucuronide and free aglycone diometin (3′,5,7-trihydroxy-4′-methoxyflavone) were first reported in lotus. Flavonoid composition varied largely with tissue type, and diverse compounds (5–15) were found in leaf and flower stalks, flower pistils, seed coats and embryos. Flower tissues including flower petals, stamens, pistils, and, especially, reproductive tissue fruit coats had more flavonoid compounds (15–17) than leaves (12), while no flavonoids were detectable in seed kernels. The flavonoid content of seed embryos was high, 730.95mg 100g−1 DW (dry weight). As regards the other tissues, mature leaf pulp (771.79mg 100g−1 FW (fresh weight)) and young leaves (650.67mg 100g−1 FW) had higher total flavonoid amount than flower stamens (359.45mg 100g−1 FW) and flower petals (342.97mg 100g−1 FW), while leaf stalks, flower stalks and seed coats had much less total flavonoid (less than 40mg 100g−1 FW).

Dissipation and Evaluation of Metaldehyde Residues in Cabbage Grown in Open Fields

The residual levels and dissipation rates of metaldehyde in cabbage and soil at three main cabbage-growing areas in china were determined by A simple，rapid and sensitive analytical method with liquid chromatography with TripleQuad mass spectrometry (LC–MS–MS). The limit of quantification (LOQ) was 0.0005,0.0005mg/kg for soil and cabbage, respectively. The mean half-life of metaldehyde was 5.84 days in cabbage and 3.17 days in soil. The final residues in cabbage and soil detected at 7 days after the last spreading were considerably all below 1mg/kg (the MRL of China). Low residues in cabbage and soil suggest that this pesticide may be safe to use under the recommended dosage.