To locate double bonds dimethyldisulfide adducts the protocol previously reported (Feng and Cronan, 2009 (link)) was used. Fatty acid methyl esters in hexane (100 μl) were converted to their dimethyldisulfide adducts by treatment with 75 μl of dimethyldisulfide and one drop of 6%, iodine solution in diethyl ether for 14 h at 50°C. Samples were cooled, and 50 μl of 10% aqueous Na2S2O3 were added to remove iodine. The hexane layer was pooled and concentrated to 50 μl under N2. Gas chromatography-mass spectroscopy analyses were done on an Agilent system consisting of a 5975C mass selective detector, a 7683B autosampler, and a 7890A gas chromatograph equipped with ZB-5MS (60 m×0.32 mm I.D. and 0.25μm film thickness) capillary column (Phenomenex, CA, USA). Injection temperature and the mass selective detector transfer line were set to 250 °C, the ion source and MS quadrupole were adjusted to 230 and 150°C, respectively. The helium carrier gas was set at a constant flow rate of 2 ml/min. The temperature program was: 2 min at 100°C, followed by an oven temperature increase of 8°C/min until 300°C. A 1 μl sample was injected with a split ratio of 10:1. The spectra acquired were recorded in the m/z 50–500 scanning range and processed using the Mass Hunter Quantitative Analysis B.08.00 (Agilent Inc., CA, USA) software.
Mass hunter quantitative analysis b 08
Manufactured by Agilent Technologies
Sourced in United States
Mass Hunter Quantitative Analysis B.08.00 is a software application developed by Agilent Technologies for data analysis and reporting of quantitative mass spectrometry experiments. The software provides tools for processing, analyzing, and reporting quantitative data obtained from Agilent mass spectrometry instruments.
Automatically generated - may contain errors
Lab products found in correlation
Zb 5ms, by Phenomenex (1 mentions)
Masshunter qualitative analysis b08, by Agilent Technologies (1 mentions)
Agilent 7890, by Agilent Technologies (1 mentions)
Hp innowax column, by Agilent Technologies (1 mentions)
Prism 8, by GraphPad (1 mentions)
5977a msd, by Agilent Technologies (1 mentions)
Zb 624, by Phenomenex (1 mentions)
Masshunter qualitative analysis navigator b 08, by Agilent Technologies (1 mentions)
7 protocols using mass hunter quantitative analysis b 08
1
Fatty Acid Dimethyldisulfide Adduct Analysis
2
Quantitative Analysis of Milk Oxylipins
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Peaks were analyzed using Agilent MassHunter Quantitative Analysis B.08.00. Peaks above a signal-to-noise ratio of 3 were manually integrated. Analyte amounts per sample were determined after correcting for the response factor of sample peak areas with an external standard curve and adjusting for extraction losses with the deuterated surrogate standards. Deuterated standard recoveries were calculated by dividing the peak area of the surrogate standards in the sample extract by the peak area of pure surrogate standards dissolved in methanol, and multiplying by 100. The analyte amount was normalized to the wet weight of cream and pellets or to the volume of skim milk. To compare oxylipin concentrations among NL-bound, PL-bound, and free oxylipins in each milk fraction, one-way analysis of variance (ANOVA) followed by Newman-Keuls test was applied per milk fraction if oxylipins were present in all three pools (PL, NL, and free). An unpaired t-test was used for oxylipins detected in two out of the three lipid pools. Data were analyzed using R v3.5.1 (https://www.Rproject.org).
3
Agilent MassHunter Data Processing Protocol
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Agilent MassHunter Qualitative Analysis B.08.00 and Agilent MassHunter Quantitative Analysis B.08.00 were used for processing of obtained raw data. For semi-quantitative analysis a minimal signal to noise ratio of 1:5 and a retention time deviation of 0.4 min were set as cut-off for peak integration. Quantifier MRM transitions were used for peak integration. Peak areas were divided by the internal standard isoguanosine. Blanks were subtracted and peak areas were further normalised by division of total peak areas of the chromatogram. Finally each variable was mean-centred and divided by the range of each variable, called range scaling33 (link)–38 (link).
4
Gas Chromatography-Mass Spectrometry of Cecum Samples
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Cecum samples (Cohort 2) were analyzed on an Agilent 7890 (Agilent Inc., Palo Alto, CA, United States) gas chromatograph, with an Agilent 5975 mass selective detector and Agilent 7683B autosampler. One microliter of sample was injected in a split mode (15:1), and analyzed on a 30 m HP-INNOWAX column with 0.25 mm inner diameter (I.D.) and 0.25 μm film thickness (Agilent, Palo Alto, CA, United States) with an injection temperature of 200°C, MSD transfer line of 200°C, and the ion source adjusted to 230°C. The helium carrier gas was set at a constant flow rate of 1 ml min–1. The temperature program was 2 min at 70°C, followed by an oven temperature ramp of 10°C min–1 to 190°C and 40°C to 240°C for a final 2 min. The mass spectrometer operated in positive electron impact mode (EI) at 69.9 eV ionization energy in m/z 30–300 scan range in combined scan and selected ion monitoring (SIM) modes. SIM targeted m/z 43, 45, 46, 60, 74. Target peaks were evaluated using Mass Hunter Quantitative Analysis B.08.00 (Agilent Inc., United States) software. Standard curves were generated for 0.1–50 mg L–1 range. At collection, an aliquot of each sample was weighed and used for dry matter (DM) analysis. Concentrations obtained from the chromatographer were then corrected for DM content and expressed as mmol g –1.
5
Quantitative Analysis of Analytical Data
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Data processing and statistical analysis were performed using Excel, GraphPad Prism 8.4.3, and R 3.6.1 (R Development Core Team, 2008 ) with the “ggplot” package (Wickham, 2016 ). Analytical data were evaluated using Agilent MassHunter Quantitative Analysis B08.00.
6
VOC Quantification by GC-MS Analysis
7
Untargeted Metabolomics Data Analysis
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Agilent MassHunter Acquisition B.08.00 (Agilent Technologies) was used for the control of the equipment and acquisition of data. MassHunter Qualitative Analysis Navigator B.08.00 and MassHunter Quantitative Analysis B.08.00 (Agilent Technologies) were used for data processing. Orthogonal partial least squares-discriminant analysis (OPLS-DA), boxplot, heatmap and receiver operating characteristic (ROC) curves were generated using MetaboAnalyst 5.0 (http://www.metaboanalyst.ca/ ). All quantitative data in this study are expressed as the mean ± standard deviation (SD). Differences were evaluated by an unpaired t-test. P < 0.05 was recognized as statistically significant.
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