scholarly journals Production of Four 15N-Labelled Cobalamins via Biosynthesis Using Propionibacterium freudenreichii

2021 ◽  
Vol 12 ◽  
Author(s):  
Mengle Wang ◽  
Stefan Asam ◽  
Jianqi Chen ◽  
Matthias Ehrmann ◽  
Michael Rychlik
Keyword(s):  
Stable Isotope ◽  
De Novo ◽  
Matrix Effects ◽  
Solid Phase ◽  
Defined Medium ◽  
Preparative Hplc ◽  
Stable Isotope Dilution ◽  
Enzyme Cofactors ◽  
Stable Growth ◽  
Sensitivity Specificity

Cobalamins (vitamin B12) are required by humans for their essential roles as enzyme cofactors in diverse metabolic processes. The four most common cobalamin vitamers are hydroxocobalamin (OHCbl), adenosylcobalamin (AdoCbl), methylcobalamin (MeCbl), and cyanocobalamin (CNCbl). Humans are not able to synthesise cobalamins de novo and thus must acquire them from external sources. Therefore, a reliable and robust analytical method to determine the cobalamins in dietary sources is highly required. For such a purpose, stable isotope dilution assays (SIDAs) with LC-MS/MS are most suited due to their superior sensitivity, specificity, and ability to compensate for matrix effects and analyte loss during sample work-up. However, a critical bottleneck for developing a SIDA method for cobalamins is the availability of stable isotope-labelled internal standards. In the present study, we harnessed the potential of Propionibacterium (P.) freudenreichii for the biosynthesis of 15N-labelled cobalamins. First, we developed a chemically defined medium (CDM) containing ammonium sulphate as a single nitrogen source except three essential vitamins that supported long-term stable growth of P. freudenreichii throughout continuous transfers. The CDM was further optimised for cobalamin production under different incubation schemes. With the optimised CDM and incubation scheme, fully 15N-labelled cobalamins were obtained in P. freudenreichii with a final yield of 312 ± 29 μg/L and 635 ± 102 μg/L, respectively, for [15N]-OHCbl and [15N]-AdoCbl. Additionally, an optimised incubation process under anaerobic conditions was successfully employed to produce specifically labelled [15N, 14N2]-cobalamins, with a yield of 96 ± 18 μg/L and 990 ± 210 μg/L, respectively, for [15N, 14N2]-OHCbl and [15N, 14N2]-AdoCbl. The labelled substances were isolated and purified by solid phase extraction and semi-preparative HPLC. Chemical modifications were carried out to produce [15N]-CNCbl and [15N]-MeCbl. Eventually, 15N-labelled compounds were obtained for the four cobalamin vitamers in high chromatographic and isotopic purity with desired 15N-enrichment and labelling patterns, which are perfectly suited for future use in SIDAs or other applications that require isotopologues.

A sensitive method to determine melatonin in saliva by automated online in-tube solid-phase microextraction coupled with stable isotope-dilution liquid chromatography-tandem mass spectrometry

2017 ◽  
Vol 9 (21) ◽  
pp. 3134-3140 ◽  
Author(s):  
Atsushi Ishizaki ◽  
Akiko Uemura ◽  
Hiroyuki Kataoka
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Stable Isotope ◽  
Solid Phase Microextraction ◽  
Solid Phase ◽  
Tandem Mass ◽  
Stable Isotope Dilution ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
Sensitive Method

Melatonin (MLT) plays important roles in regulating the sleep-wake cycle, and has many beneficial effects on health. A simple, rapid, and sensitive method was developed for the determination of MLT in human saliva by automated online in-tube solid-phase microextraction coupled with stable isotope-dilution liquid chromatography-tandem mass spectrometry.

scholarly journals Evaluation of a Stable Isotope-Based Direct Quantification Method for Dicamba Analysis from Air and Water Using Single-Quadrupole LC–MS

Molecules ◽  
2020 ◽  
Vol 25 (16) ◽  
pp. 3649 ◽  
Author(s):  
Manoj Ghaste ◽  
Nicholas C. Hayden ◽  
Matthew J. Osterholt ◽  
Julie Young ◽  
Bryan Young ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Water Samples ◽  
Detection Method ◽  
Matrix Effects ◽  
Solid Phase ◽  
Internal Standard ◽  
Sample Collection ◽  
Selected Ion Monitoring ◽  
Ms Detection ◽  
Direct Quantification

Dicamba is a moderately volatile herbicide used for post-emergent control of broadleaf weeds in corn, soybean, and a number of other crops. With increased use of dicamba due to the release of dicamba-resistant cotton and soybean varieties, growing controversy over the effects of spray drift and volatilization on non-target crops has increased the need for quantifying dicamba collected from water and air sampling. Therefore, this study was designed to evaluate stable isotope-based direct quantification of dicamba from air and water samples using single-quadrupole liquid chromatography–mass spectrometry (LC–MS). The sample preparation protocols developed in this study utilize a simple solid-phase extraction (SPE) protocol for water samples and a single-step concentration protocol for air samples. The LC–MS detection method achieves sensitive detection of dicamba based on selected ion monitoring (SIM) of precursor and fragment ions and relies on the use of an isotopically labeled internal standard (IS) (D3-dicamba), which allows for calculating recoveries and quantification using a relative response factor (RRF). Analyte recoveries of 106–128% from water and 88–124% from air were attained, with limits of detection (LODs) of 0.1 ng mL−1 and 1 ng mL−1, respectively. The LC–MS detection method does not require sample pretreatment such as ion-pairing or derivatization to achieve sensitivity. Moreover, this study reveals matrix effects associated with sorbent resin used in air sample collection and demonstrates how the use of an isotopically labeled IS with RRF-based analysis can account for ion suppression. The LC–MS method is easily transferrable and offers a robust alternative to methods relying on more expensive tandem LC–MS/MS-based options.

scholarly journals The Pear Aroma in the Austrian Pinot Blanc Wine Variety: Evaluation by Means of Sensorial-Analytical-Typograms with regard to Vintage, Wine Styles, and Origin of Wines

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Christian Philipp ◽  
Phillip Eder ◽  
Walter Brandes ◽  
Elsa Patzl-Fischerleitner ◽  
Reinhard Eder
Keyword(s):  
Gas Chromatography ◽  
Stable Isotope ◽  
Solid Phase Microextraction ◽  
Solid Phase ◽  
Stable Isotope Dilution ◽  
Isotope Dilution Assay ◽  
Dilution Assay ◽  
Consumer Study ◽  
Gas Chromatography Mass Spectroscopy ◽  
Chain Ester

The perceived pear aroma in wines was associated with various short- and medium-chain ester compounds. A consumer study confirmed this assumption. In total, eight ester compounds from a series of 100 aromatic substances were associated with the pear aroma. In this study, a valid stable isotope dilution assay headspace solid-phase microextraction gas chromatography mass spectroscopy (SIDA-HS-SPME-GC-MS) method was developed for the analysis of these compounds, and 102 Austrian Pinot blanc wines of vintages 2013–2016 were analysed. They were assessed with regard to vintage and origin influences as well as wine styles. However, an attempt was made to capture the synergies of these compounds for the pear aroma. With the detection of ethyl (E,Z)-2,4-decadienoate and methyl (E)-geranoate, two volatile compounds were measured which had not previously been detected in Austrian wines. The eight analysed esters were perceived very differently. Therefore, specific odour activity values for the various sensations could be calculated with a mathematical combination of the analyses and the results of the sensory studies. A vintage influence on the sensorial descriptor “overripe pear” and a relationship between wine style and total pear aroma were determined.

scholarly journals Quantification of α-Thujone and Its Metabolites in Human Urine after Consumption of a Sage Infusion Using Stable Isotope Dilution Assays

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 511 ◽  
Author(s):  
Irene Thamm ◽  
Konrad Tiefenbacher ◽  
Michael Rychlik
Keyword(s):  
Detection Limit ◽  
Stable Isotope ◽  
Isotope Dilution ◽  
Human Urine ◽  
Maximum Concentration ◽  
Solid Phase Microextraction ◽  
Solid Phase ◽  
Internal Standard ◽  
Stable Isotope Dilution ◽  
Stable Isotope Dilution Assays

The metabolism of the monoterpene α-thujone was investigated in humans after consumption of sage tea, by analyses of its metabolites 2-hydroxythujone, 4-hydroxythujone, and 7-hydroxythujone in urine. For the quantitation of α-thujone and its metabolites, stable isotope dilution assays were developed. Using d6-α-thujone as internal standard, we quantified α-thujone by solid phase microextraction GC-MS and the hydroxythujones with d6-2-hydroxythujone, d6-4-hydroxythujone, and d6-7-hydroxythujone after glucuronide/sulfate deconjugation by LC-MS/MS in urine. After the consumption of 575.0 µg α-thujone, the 4-hydroxythujone and 7-hydroxythujone were detectable in the urine of the volunteers under study, after liberation from their glucuronides/sulfates. The 2-Hydroxythujone was not present in any of the volunteer samples above its detection limit. α-Thujone was detectable in a low amount, with a maximum concentration of 94 ng/L for the volunteer with the highest dose of 14.3 µg/kg bw.

Application of Stable Isotope Dilution and Liquid Chromatography Tandem Mass Spectrometry for Multi-Mycotoxin Analysis in Edible Oils

2019 ◽  
Vol 102 (6) ◽  
pp. 1651-1656
Author(s):  
Kai Zhang ◽  
David Xu
Keyword(s):  
Stable Isotope ◽  
Isotope Dilution ◽  
Corn Oil ◽  
Edible Oils ◽  
Matrix Effects ◽  
Safety Issue ◽  
Response Factors ◽  
Calibration Standards ◽  
Stable Isotope Dilution ◽  
Accuracy And Repeatability

Background: Mycotoxin contamination in oils remains an important food safety issue. To monitor the occurrence of mycotoxins in edible oils, it is important to develop analytical methods that can determine multiple mycotoxins in oil products. A stable isotope dilution LC-tandem MS (LC-MS/MS) method for the simultaneous determination of 12 mycotoxins in five edible oil matrixes (canola, corn, olive, peanut, and soybean oil) was developed and validated. Methods: Prior to extraction, the oil samples were fortified with ¹³C uniformly labeled internal standards (¹³C-IS) for 12 target mycotoxins, followed by extraction and LC-MS/MS analysis. Quantitation was achieved using solvent-only calibration standards, relative response factors of ¹³C-IS, and target mycotoxins. Results: The majority of recoveries in oil for ochratoxin A and aflatoxins B1, B2, G1, and G2 fortified at 1, 10, and 100 ng/g as well as deoxynivalenol; fumonisins B1, B2, and B3; T-2 toxin; HT-2 toxin; and zearalenone fortified at 10, 100, and 1000 ng/g ranged from 80 to 120% with RSDs of <20%. The method LOQs ranged from 0.1 ng/g (aflatoxin B1) to 6.4 ng/g (zearalenone). Among 16 U.S. market samples, zearalenone was detected in three corn oil samples at 37, 185, and 317 ng/g, respectively. T-2 toxin was found in two corn oil samples at 7 and 10 ng/g, respectively. Conclusions: The method provides sufficient selectivity, sensitivity, accuracy, and repeatability to screen edible oils for regulated mycotoxins such as aflatoxins at low nanogram per gram concentrations without using conventional standard addition or matrix-matched calibration standards to correct for matrix effects.

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