We used an Agilent Technologies 1290 high performance liquid chromatography (HPLC) system coupled with an Agilent 6470 Triple Quadrupole liquid chromatography tandem mass spectrometry (LC/MS, Wilmington, DE, USA) to determine plasma levels of H2S and thiosulfate as described previously [18 (link)]. The H2S derivative sulfide dibimane (SDB) and thiosulfate derivative pentafluorobenzyl (PFB)-S2O3H were determined. The detection of target compounds was conducted in the selected reaction monitoring mode using transitions of m/z 415→223, m/z 292.99→81, and m/z 212.99→93, for SDB, PFB-S2O3H, and PHB, respectively. We used phenyl 4-hydroxybenzoate (PHB) as an internal standard.
6470 triple quadrupole
Manufactured by Agilent Technologies
Sourced in Germany
The 6470 triple quadrupole is a high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) system designed for analytical applications. It features a triple quadrupole mass analyzer configuration to provide accurate and sensitive quantitative analysis of chemical compounds.
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Lab products found in correlation
1290 uhplc system, by Agilent Technologies (2 mentions)
Mass hunter quantitative software, by Agilent Technologies (2 mentions)
Eclipse plus c18 column, by Agilent Technologies (1 mentions)
1290 infinity 2 lc system, by Agilent Technologies (1 mentions)
1290 infinity 2, by Agilent Technologies (1 mentions)
1260 infinity 2, by Agilent Technologies (1 mentions)
Cortisol ria kit, by Beckman Coulter (1 mentions)
Orbitrap fusion lumos tribrid, by Thermo Fisher Scientific (1 mentions)
Lysing matrix e tube, by MP Biomedicals (1 mentions)
Tsq quantiva, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna stool mini kit protocol, by Qiagen (1 mentions)
Precellys 24 homogenizer, by Bertin Technologies (1 mentions)
Heptafluorobutyric acid hfba, by Merck Group (1 mentions)
Kinetex c18 column, by Phenomenex (1 mentions)
7 protocols using 6470 triple quadrupole
1
Quantification of Plasma H2S and Thiosulfate
2
Multianalyte LC-MS/MS Quantification
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For the first, second and third groups of substances, an Agilent 1290 Infinity II LC System coupled with an Agilent 6470 Triple Quadrupole was used. The chromatography (classical and SFPE procedures) was performed with the Zorbax Eclipse Plus C18 column (RRHD 2.1 × 50 mm, 1.8 μm). The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (100% acetonitrile). The gradient elution was conducted as follows: 0 min: 95% A, 5% B; 8 min: 5% A, 95% B, for the first and second groups of substances; the analysis run time was 8 min. Conversely, for the third group, it was as follows: 0 min: 95% A, 5% B; 6.50 min: 60% A, 40% B; 6.51 min: 5% A, 95% B; 8 min: 5% A, 95% B; the analysis run time was 8 min. The flow rate was 0.4 mL/min. MS data were obtained in the positive ion modes (multiple reaction monitoring mode) with electrospray probe voltages of 4000 V. The nebulizer gas setting was 45 psi for the first group of substances and 40 psi for the second and third. The ion source was operated at a temperature of 350 °C and a drying gas setting of 12 L/min for the first and third group substances, and 250 °C, 10 L/min for the second group. The MRM (multiple reaction monitoring) transitions for each group of substances are shown in Table 7 .
3
PFAS Quantification by LC-MS/MS
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Instrumental analysis was completed according to Li et al. [17 (link)]. PFAS analyses were conducted using an Agilent 1290 Infinity II liquid chromatograph coupled with an Agilent 6470 triple quadrupole (LC-MS/MS) equipped with a jet stream electrospray ionization source. PFAS free tubing and a delay column (Hypersil GOLD aQ C18, 20 × 2.1 mm, 12 μm) placed between the mobile phase mixer and the sample injector were used in the LC to avoid possible contamination. A Hypersil GOLD pentafluorophenyl (PFP) column (150 × 2.1 mm, 3 μm) fitted with a PFP guard column (Hypersil Gold PFP 5 μm drop-in guards) was used to separate PFAS analogues. The temperature and the flow rate were set at 50 °C and 0.4 mL min−1, respectively. The mobile phase used was methanol and 5 mM ammonium formate in LC-MS water and injection volume was 100 µL for water sample extracts and 20 µL for plant and sediment extracts. Tables S2 and S3 (Supplementary Material) show the LC gradient conditions, and the MS parameters, respectively. A dynamic multiple-reaction monitoring (MRM) technique in negative mode was used for sample acquisition and determination of multiple PFAS. Table S4 (Supplementary Material) shows in detail the MRM method, which includes data on precursor and product ions that were monitored, their retention time, fragmentor voltage, and collision energy.
4
Plasma Cortisol, UFC, and ACTH Measurement
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Measurement of plasma cortisol was performed using Siemens Atellica IM Cortisol kit (intra-assay variation coefficient ≤ 2.3% for plasma, inter-assay variation coefficient ≤ 7.7% for plasma). Reported cross-reactivity with steroid precursors: 11-deoxycortisol ≤ 25%, 21-deoxycortisol ≤ 15%, 17α-hydroxyprogesterone ≤ 2%, pregnenolone ≤ 0,5%, and progesterone ≤ 1%.
The 24 h UFC was measured by a Beckman Coulter Cortisol RIA kit (intra-assay variation coefficient ≤ 8.9% for urine, inter-assay variation coefficient ≤ 13.3% for urine). Blood samples for ACTH measurement were kept on ice, temporarily frozen at −20°C and analysed with a Thermo Scientific BRAHMS ACTH RIA kit (intra-assay variation coefficient 3.5%, inter-assay variation coefficient 4.7%).
Plasma steroids including cortisol were measured by two-dimensional liquid chromatography–tandem mass spectrometry (Agilent technologies 1260 Infinity II, Agilent 6470 triple quadrupole) using pre-mixed sets of steroids and their internal standards (Chromsystems Instruments & Chemicals GmbH) with in-house developed method for a multiplex quantitative analysis of steroid hormones (3 (link)).
The 24 h UFC was measured by a Beckman Coulter Cortisol RIA kit (intra-assay variation coefficient ≤ 8.9% for urine, inter-assay variation coefficient ≤ 13.3% for urine). Blood samples for ACTH measurement were kept on ice, temporarily frozen at −20°C and analysed with a Thermo Scientific BRAHMS ACTH RIA kit (intra-assay variation coefficient 3.5%, inter-assay variation coefficient 4.7%).
Plasma steroids including cortisol were measured by two-dimensional liquid chromatography–tandem mass spectrometry (Agilent technologies 1260 Infinity II, Agilent 6470 triple quadrupole) using pre-mixed sets of steroids and their internal standards (Chromsystems Instruments & Chemicals GmbH) with in-house developed method for a multiplex quantitative analysis of steroid hormones (3 (link)).
5
Standardized Gut Microbiome Analysis
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DNA of colonic biopsies and stool samples was extracted using the standard QIAamp DNA stool mini kit protocol (Qiagen) modified by an initial bead-beating-step with Lysing Matrix E tubes (MP Biomedicals) and a Precellys 24 homogenizer (Bertin instruments) with 5200 rpm 3 × 30 seconds for colonic biopsies and 5500 rpm 1 × 30 seconds for stool samples. Bacterial 16S rRNA gene copy number was quantified using quantitative polymerase chain reaction and normalized to the total amount of double-stranded DNA (assessed with a Quant-iT PicoGreen dsDNA Assay Kit). Metabolomics was performed on a subset of ileal biopsies (5 BF+ biopsies and 5 BF– biopsies) using liquid chromatography–mass spectrometry
(Orbitrap Fusion Lumos Tribrid; Thermo Fisher Scientific) for lipid analysis and liquid chromatography–tandem mass spectrometry (6470 triple quadrupole; Agilent Technologies) for metabolite analysis. BA composition of stool samples was analyzed using liquid chromatography–tandem mass spectrometry (TSQ Quantiva; Thermo Fisher Scientific). Methodology, sample numbers, and bioinformatics workflow are described in more detail in theSupplementary Methods and Supplementary Figure 3 .
(Orbitrap Fusion Lumos Tribrid; Thermo Fisher Scientific) for lipid analysis and liquid chromatography–tandem mass spectrometry (6470 triple quadrupole; Agilent Technologies) for metabolite analysis. BA composition of stool samples was analyzed using liquid chromatography–tandem mass spectrometry (TSQ Quantiva; Thermo Fisher Scientific). Methodology, sample numbers, and bioinformatics workflow are described in more detail in the
6
UHPLC-MS/MS Targeted Metabolite Analysis
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Targeted analysis was performed on a UHPLC 1290 system (Agilent Technologies, Waldbronn, Germany) coupled to a Triple Quadrupole 6470 (Agilent Technologies) equipped with an electrospray source operating in negative mode. The gas temperature was set to 300° C with a gas flow of 12 l/min. The capillary voltage was set to 5000 kV.
10 μL of sample were injected on a Column Zorbax Eclipse XBD C18 (100 mm x 2.1 mm particle size 1.8 μm) from Agilent technologies, protected by a guard column XDB-C18 (5 mm × 2.1 mm particle size 1.8 μm) and heated at 50° C by a Pelletier oven.
Gradient mobile phase consisted of water with 0.01% of formic acid (A) and acetonitrile with 0.01% of formic acid (B). Flow rate was set to 0.4 mL/min, and gradient as follow: initial condition was 80% phase A and 20% phase B, maintained during 6 min. Molecules were then eluted using a gradient from 20% to 45% phase B over 7 min. Column was washed using 95% mobile phase B for 5 minutes and equilibrated using 20% mobile phase B for 4 min. Autosampler was kept at 4° C.
The collision gas was nitrogen. The scan mode used was the MRM for biological samples. Peak detection and integration of analytes were performed using the Agilent Mass Hunter quantitative software (B.07.01), exported as tables and processed with R software (version 4.0.3) and the GRMeta package (Github/kroemerlab).
10 μL of sample were injected on a Column Zorbax Eclipse XBD C18 (100 mm x 2.1 mm particle size 1.8 μm) from Agilent technologies, protected by a guard column XDB-C18 (5 mm × 2.1 mm particle size 1.8 μm) and heated at 50° C by a Pelletier oven.
Gradient mobile phase consisted of water with 0.01% of formic acid (A) and acetonitrile with 0.01% of formic acid (B). Flow rate was set to 0.4 mL/min, and gradient as follow: initial condition was 80% phase A and 20% phase B, maintained during 6 min. Molecules were then eluted using a gradient from 20% to 45% phase B over 7 min. Column was washed using 95% mobile phase B for 5 minutes and equilibrated using 20% mobile phase B for 4 min. Autosampler was kept at 4° C.
The collision gas was nitrogen. The scan mode used was the MRM for biological samples. Peak detection and integration of analytes were performed using the Agilent Mass Hunter quantitative software (B.07.01), exported as tables and processed with R software (version 4.0.3) and the GRMeta package (Github/kroemerlab).
7
UHPLC-MS/MS Quantification of Analytes
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Targeted analysis was performed on a UHPLC 1290 system (Agilent Technologies, Waldbronn, Germany) coupled to a Triple Quadrupole 6470 (Agilent Technologies) equipped with an electrospray source operating in positive mode. The gas temperature was set to 350° C with a gas flow of 12 l/min. The capillary voltage was set to 2.5 kV.
10 μL of sample were injected on a Column Kinetex C18 (150 mm x 2.1 mm particle size 2.6 μm) from Phenomenex, protected by a guard column C18 (5 mm × 2.1 mm) and heated at 40° C in a Pelletier oven.
The gradient mobile phase consisted of water with 0.1 % of Heptafluorobutyric acid (HFBA, Sigma-Aldrich) (A) and acetonitrile with 0.1 % of HFBA (B) freshly made. The flow rate was set to 0.2 ml/min, and gradient as follow: initial condition was 95% phase A and 5% phase B. Molecules were then eluted using a gradient from 5% to 40% phase B over 10 min. The column was washed using 90% mobile phase B for 2.5 minutes and equilibrated using 5% mobile phase B for 4 min. The autosampler was kept at 4° C.
The collision gas was nitrogen. The scan mode used was the MRM for biological samples. Peak detection and integration of analytes were performed using the Agilent Mass Hunter quantitative software (B.07.01), exported as tables and processed with R software (version 4.0.3) and the GRMeta package (Github/kroemerlab).
10 μL of sample were injected on a Column Kinetex C18 (150 mm x 2.1 mm particle size 2.6 μm) from Phenomenex, protected by a guard column C18 (5 mm × 2.1 mm) and heated at 40° C in a Pelletier oven.
The gradient mobile phase consisted of water with 0.1 % of Heptafluorobutyric acid (HFBA, Sigma-Aldrich) (A) and acetonitrile with 0.1 % of HFBA (B) freshly made. The flow rate was set to 0.2 ml/min, and gradient as follow: initial condition was 95% phase A and 5% phase B. Molecules were then eluted using a gradient from 5% to 40% phase B over 10 min. The column was washed using 90% mobile phase B for 2.5 minutes and equilibrated using 5% mobile phase B for 4 min. The autosampler was kept at 4° C.
The collision gas was nitrogen. The scan mode used was the MRM for biological samples. Peak detection and integration of analytes were performed using the Agilent Mass Hunter quantitative software (B.07.01), exported as tables and processed with R software (version 4.0.3) and the GRMeta package (Github/kroemerlab).
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