1290 infinity uhplc

BPA and DEHP metabolites (MEHP, 5OH-MEHP, and 5oxo-MEHP) levels are determined on urine samples in subjects from HBM and case-control studies. Upon arrival to CNR laboratory, samples are checked, catalogued, and aliquoted for sample analysis and storage in the biobank. As phthalates and BPA are metabolized and excreted in the urine in a glucuronidated form (that is soluble in water, while phthalates and BPA are hydrophobic); the analysis requires a previous enzymatic deconjugation (β-glucuronidase enzyme from Helix pomatia, ≥ 100.000 units/mL Sigma Aldrich) to hydrolyze the glucuronide form and separate free phthalate metabolites and BPA. Samples are purified through individual SPE cartridge C18 ODS 3 ml tubes 200 mg (Agilent), and then analyzed by LC–ESI-MS QTOF (Agilent UHPLC 1290 infinity coupled with Agilent 6540 Quadrupole Time-of-Flight (QTOF) system equip with Agilent ZORBAX SB-Phenyl 2.1x100mm 1.8-Micron), for DEHP’s metabolites and by GC/MS (Agilent GC-7890 coupled with Agilent MS-5975C) for BPA quantification. Since samples are from spot urine, they need to be normalized with respect to creatinine concentration. For each subject, creatinine concentrations were measured by AU400 Beckman analyzer using an enzymatic method (Beckman Coulter).

A JASCO P-2000 digital polarimeter was utilized to measure the specific rotations of the compounds. A Bruker DRX-400 NMR spectrometer was utilized to obtain the NMR spectra of the compounds. Specifications of the NMR spectrometer include a Cryoprobe, and a 5 mm BBI (1H, G-COSY, multiplicity-edited G-HSQC, and G-HMBC spectra) or BBO (13C spectra) probe heads equipped with z-gradients. Residual solvent peaks for DMSO-d₆ were set at δ_H 2.50 and δ_C 39.5 ppm as reference signals in the ¹H and ¹³C NMR spectra, respectively. A preparative HPLC experiment was performed using Agilent 1260 Infinity Preparative-scale LC/MS Purification System coupled to an Agilent 6130B single quadrupole mass spectrometer with an XTerra Prep MS C₁₈ column (19 × 300 mm, 10 µm). The detection wavelength used in the preparative HPLC was 254 nm. An Agilent UHPLC 1290 Infinity, coupled with an Agilent 6540 accurate–mass quadrupole time-of-flight (QTOF) mass spectrometer, equipped with an ESI source was utilized to conduct the HPLC-MS experiment. The analyses were conducted with an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm), at a flow rate of 0.5 mL/min under standard gradient conditions of 2% MeCN (0.1% formic acid) to 100% MeCN (0.1% formic acid) over 8.6 min. The operating parameters for QTOF were the same as previously reported [8 (link)].

For targeted metabolomic analysis, samples were precipitated and analyzed using a UHPLC 1290 Infinity instrument (Agilent Technologies, USA) and a coupled quadrupole time-of-flight mass spectrometer (AB TripleTOF 6600) equipped with an electrospray ionization source. Detection was performed in both negative and positive ion modes. The raw MS data were converted into MzXML files using a ProteoWizard MSConvert tool and processed using XCMS for feature detection, retention time correction, and alignment. The metabolites were identified by accuracy mass spectrometry (<25 ppm) and MS/MS data, which matched our standard database. For multivariate statistical analysis, the web-based MetaboAnalyst system (https://www.metaboanalyst.ca/) was used, followed by Pareto scaling, PCA, and OPLS-DA. The significantly different metabolites were determined based on the combination of a statistically significant threshold VIP value obtained from the OPLS-DA model and two-tailed Student’s t test (P value) on the raw data, and the metabolites with VIP values of >1.0 and P values of <0.05 were considered significant.

The concentration of coupled peptide was quantitated as previously described [26 (link)]. The amount of VCAM-1 recognizing peptide attached to the surface of Nar/LN was measured indirectly by detecting the amount of peptide that remained uncoupled after purification on the Amicon Ultra 100 KDa column. UHPLC measurements were done with an Agilent Technologies UHPLC 1290 Infinity instrument equipped with a binary pump, autosampler, column oven, and diode array UV/VIS detector. Separation was performed on an Eclipse Plus ZORBAX C18 column narrow bore RR (150 × 2.1 mm, 3.5 µm) with the column oven temperature kept at 25 °C. The mobile phase consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B) with a gradient elution of 5% B (0–0.8 min), 5–26% B (0.8–8 min), 26% B (8–10 min), and 26–5% B (10–12 min), at a flow rate of 0.25 mL/min. The detection was performed at the wavelength 220 nm. System control and data analysis were carried out using Agilent ChemStation software (B.04.02 Version, Agilent Technologies, Santa Clara, CA, USA).

Optical rotations were recorded on a JASCO P-2000 digital polarimeter. UV spectra were obtained on a GE Healthcare Ultrospec 9000 spectrophotometer. NMR spectra were collected on a Bruker DRX-400 NMR spectrometer with Cryoprobe, using 5-mm BBI (¹H, G-COSY, multiplicity-edited G-HSQC, and G-HMBC spectra) or BBO (¹³C spectra) probe heads equipped with z-gradients. Spectra were calibrated to residual protonated solvent signals (CD₃OD δ_H 3.30 and CD₃OD δ_C 49.0; DMSO-d₆ δ_H 2.49 and DMSO-d₆ δ_C 39.5). Preparative HPLC analysis was performed on the Agilent 1260 Infinity Preparative-Scale LC/MS Purification System, completed with Agilent 6130B single quadrupole mass spectrometer for LC and LC/MS Systems. The samples were separated on an Agilent Prep C18 column (100 × 30 mm) by gradient elution with a mixture of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The HR-ESI-MS spectra were acquired on Agilent UHPLC 1290 Infinity coupled to Agilent 6540 accurate-mass quadrupole time-of-flight (QTOF) mass spectrometer equipped with a splitter and an ESI source. The analysis was performed with a C18 4.6 × 75 mm, 2.7 µm column at flowrate of 2 mL/min, under standard gradient condition of 0.1% formic acid in water and 0.1% formic acid in acetonitrile over 15 min.