Nucleodur sphinx rp column

Comparative HPLC-MS (ion trap) analyses of aqueous feces extracts were carried out on an Agilent 1100 series HPLC (Agilent Technologies, Waldbronn, Germany) coupled to an Esquire 6000 ESI-Ion Trap mass spectrometer (Bruker Daltonics, Bremen, Germany) operated in alternating ionization mode in the range m/z 55–1000. Skimmer voltage −40 eV, capillary exit voltage −102.3 eV, capillary voltage 4,000 V, nebulizer pressure 35 psi, drying gas, 11 l/min; gas temperature 330°C. MS² and MS³ spectra of candidate metabolites were obtained using the AutoMS mode of the software (Bruker Daltonics). Separation was accomplished using a Nucleodur Sphinx RP column (250 × 4.6 mm, 5 μm particle size, Macherey-Nagel) with a mobile phase consisting of 0.2% (v/v) formic acid in ultrapure water as solvent A and acetonitrile as solvent B with a flow rate of 1.0 ml/min at 25°C. The gradient was as follows: 0–100% (v/v) B (25 min), 100% B (3 min), 100–0% (v/v) B (0.1 min), 0% B (4.9 min). Eluent flow rate was diverted in a ratio of 4:1 before entering the ESI unit. Chromatograms were analyzed with the DataAnalysis and MetaboliteTools software packages from Bruker Daltonics. Candidate metabolites were further analyzed using MS/MS and high resolution mass spectrometry (see section UHPLC-ESI-MS/MS).

Insect eye samples of around 10 mg were extracted with 0.5 mL of methanol. The samples were homogenized with the help of 3 mm stainless steel balls and a paint shaker (Skandex SO-10M, Fluid Management Europe, The Netherlands). After centrifugation an aliquot of the supernatant was analyzed by LC-ESI-IonTrap-MS using a Bruker Esquire 6000 ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) operated in alternating ionization mode (electrospray ionization) in the range m/z 60–1,000 (Skimmer voltage, 53 eV; capillary exit voltage, −117.3 eV; capillary voltage, 4,000 V; nebulizer pressure, 35 psi; drying gas, 11 l min −1; gas temperature, 330 °C) coupled to an Agilent 1100 series HPLC (Agilent Technologies, Waldbronn, Germany). Elution was accomplished using a Nucleodur Sphinx RP column (250 × 4.6 mm, 5 um; Macherey-Nagel, Duren, Germany). Mobile phases were 0.2% formic acid (v:v) (A) and acetonitrile (B), starting with 10% B, followed by a gradient to 90% B in 20 min, the column was washed with 100% B for 3 min and equilibrated for 4 min at 10%B. The molecular ion peaks [M+H]⁺ of the analytes were monitored in positive mode (extracted ion chromatograms), and the area under the peak was used for relative quantification. The peak area for each compound was normalized by the sample weight for each sample to obtain relative sample concentrations in peak area/mg of weight.

Samples of the enzyme assays were analyzed using a Waters Acquity UPLC system connected to a ThermoFisher Exactive Orbitrap high resolution mass spectrometer fitted with a heated electrospray ion source (HESI). The mass spectrometer was operated in positive ionization mode at 50000 resolution with internal calibration to the lock mass 455.12002. For UPLC separation a Macherey Nagel Nucleodur Sphinx RP column (100 × 2 mm, 1.8 μm) was used. HPLC program: 2% B 0 min, in 5 min to 100% B, 3 min 100% B, in 0.5 min to 2% B, 2.5 min 2% B, solvent A: H₂O 0.1% AcOH, solvent B: MeOH 0.1% AcOH, flow rate: 0.3 ml/min. 0.2–1 ul of the samples was injected. Aniline: 1.5 min, [M+H]⁺ m/z 94.06525, Δppm 1.3, C₆H₈N; phenylphosphoamidate: 4.0 min, [M+H]⁺ m/z 174.03140, Δppm 0.32, C₆H₉O₃NP; 4-aminobenzoate: 2.9 min, [M+H]⁺ m/z 138.05498, Δppm 0.18, C₇H₈O₂N, phenylphosphate: 4.5 min, [M+H]⁺ m/z 175.01553, C₆H₈O₄P, Δppm 0.33.